Light emission modifiers and their uses in nucleic acid detection, amplification and analysis

ABSTRACT

The present invention relates to methods and reagents for modifying the emission of light from labeled nucleic acids for the purpose of real time detection, analysis, and quantitation of nucleic acid sequences, e.g., using singly labeled probes. These methods and reagents exploit advantageous properties of thiazine dyes and diazine dyes. Furthermore, the use of these light emission modifiers in background reduction, nucleic acid duplex stabilization and other uses is also described. Related kits, reaction mixtures and integrated systems are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of a pending application Ser. No.11/474,062, filed on Jun. 23, 2006, which claims priority to and benefitof the following United States Provisional Patent Applications:Application Ser. No. 60/695,991, filed Jun. 30, 2005; Application Ser.No. 60/696,253, filed Jun. 30, 2005; Application Ser. No. 60/696,293,filed Jun. 30, 2005; and Application Ser. No. 60/696,303, filed Jun. 30,2005.

FIELD OF THE INVENTION

The present invention relates to the fields of molecular biology andnucleic acid chemistry. In certain embodiments, methods and reagents formodifying the emission of light from labeled nucleic acids are providedfor the purpose of real time homogeneous detection, analysis, andquantitation of nucleic acid sequences using singly labeled probes.Furthermore, the use of these light emission modifiers in backgroundreduction and other uses is also described.

BACKGROUND OF THE INVENTION

The development of nucleic acid amplification technology (NAT) hasrevolutionized genetic analysis and engineering science. For example,the polymerase chain reaction (PCR) is commonly utilized to amplifyspecific target nucleic acids using selected primer nucleic acids, e.g.,to facilitate the detection of the target nucleic acid as part of adiagnostic, forensic, or other application. Primers typically functionin pairs that are designed for extension towards each other to cover theselected target region. A typical PCR cycle includes a high temperature(e.g., 85° C. or more) denaturation step during which the strands ofdouble-stranded nucleic acids separate from one another, a lowtemperature (e.g., 45-65° C.) annealing step during which the primershybridize to the separated single strands, and an intermediatetemperature (e.g., around 72° C.) extension step during which a nucleicacid polymerase extends the primers. Two-temperature thermocyclingprocedures are also utilized. These generally include a high temperaturedenaturation step and a low temperature anneal-extend step. To produce adetectable amount of the particular PCR product or amplicon, thesecycles are generally repeated between about 25-45 times.

PCRs are also described in many different U.S. patents including, e.g.,U.S. Pat. No. 4,683,195, entitled “PROCESS FOR AMPLIFYING, DETECTING,AND/OR-CLONING NUCLEIC ACID SEQUENCES,” which issued to Mullis et al.Jul. 28, 1987, U.S. Pat. No. 4,683,202, entitled “PROCESS FOR AMPLIFYINGNUCLEIC ACID SEQUENCES,” which issued to Mullis Jul. 28, 1987, and U.S.Pat. No. 4,965,188, entitled “PROCESS FOR AMPLIFYING, DETECTING, AND/ORCLONING NUCLEIC ACID SEQUENCES USING A THERMOSTABLE ENZYME,” whichissued to Mullis et al. Oct. 23, 1990, which are each incorporated byreference. Further, PCR-related techniques are also described in variousother publications, such as Innis et al. (Eds.) PCR Protocols: A Guideto Methods and Applications, Elsevier Science & Technology Books (1990),Innis et al. (Eds.) PCR Applications: Protocols for Functional Genomics,Academic Press (1999), Edwards et al., Real-Time PCR, Taylor & Francis,Inc. (2004), and Rapley et al., Molecular Analysis and Genome Discovery,John Wiley & Sons, Inc. (2004), which are each incorporated byreference.

Many variations of the PCR as well as other nucleic acid amplificationtechniques have also been developed. Examples of these includereverse-transcription PCR (RT-PCR) (Joyce (2002) “Quantitative RT-PCR. Areview of current methodologies” Methods Mol. Biol. 193:83-92 and Emrichet al. (2002) “Quantitative detection of telomerase components byreal-time, online RT-PCR analysis with the LightCycler,” Methods Mol.Biol. 191:99-108), the ligase chain reaction (LCR) (Lee (1996) “Ligasechain reaction,” Biologicals 24(3):197-9), the polymerase ligase chainreaction (Barany et al. (1991) “The ligase chain reaction in a PCRworld,” PCR Methods Appl. 1(1):5-16), the Gap-LCR (Abravaya et al.(1995) “Detection of point mutations with a modified ligase chainreaction (Gap-LCR),” Nucleic Acids Res. 23(4):675-82), stranddisplacement amplification (Walker (1993) “Empirical aspects of stranddisplacement amplification,” PCR Methods Appl. 3(1):1-6), linked linearamplification (LLA) (Killeen et al. (2003) “Linked linear amplificationfor simultaneous analysis of the two most common hemochromatosismutations,” Clin Chem. 49(7):1050-7), rolling circle amplification (RCA)(Nilsson et al. (2002) “Real-time monitoring of rolling-circleamplification using a modified molecular beacon design,” Nucleic AcidsRes. 30(14):e66), transcription-mediated amplification (TMA) (Emery etal. (2000) “Evaluation of performance of the Gen-Probe humanimmunodeficiency virus type 1 viral load assay using primary subtype A,C, and D isolates from Kenya,” J Clin Microbiol 38:2688-2695),nucleic-acid-sequence-based amplification (NASBA) (Mani et al. (1999)“Plasma RNA viral load as measured by the branched DNA and nucleic acidsequence-based amplification assays of HIV-1,” J Acquir Immune DeficSyndr 22:208-209 and Berndt et al. (2000) “Comparison between a nucleicacid sequence-based amplification and branched DNA test for quantifyingHIV RNA load in blood plasma,” J Virol Methods 89:177-181), andself-sustaining sequence replication (3SR) (Mueller et al. (1997)“Self-sustained sequence replication (3SR): an alternative to PCR,”Histochem Cell Biol 108:431-7), which are each incorporated byreference.

Various strategies for detecting amplification products have beendeveloped, including those involving 5′ nuclease probes, molecularbeacons, or SCORPION® primers, among many others. To illustrate, a 5′nuclease assay typically utilizes the 5′ to 3′ nuclease activity ofcertain DNA polymerases to cleave 5′ nuclease probes during the courseof a polymerase chain reaction (PCR). These assays allow for both theamplification of a target and the release of labels for detection,generally without resort to multiple handling steps of amplifiedproducts. Certain 5′ nuclease probes include labeling moieties, such asa fluorescent reporter dye and a quencher dye. When the probe is intact,the proximity of the reporter dye to the quencher dye generally resultsin the suppression of the reporter fluorescence. In many cases, however,an intact probe produces a certain amount of residual or baselinefluorescence. During a 5′ nuclease reaction, cleavage of the probeseparates the reporter dye and the quencher dye from one another,resulting in a detectable increase in fluorescence from the reporter.The accumulation of PCR products or amplicons is typically detectedindirectly by monitoring this increase in fluorescence in real-time.

Although many pre-existing nucleic acid amplification detection formatsare simple and robust, certain challenges remain. For example, many ofthese detection formats utilize dual labeled probes (e.g., a probe thatincludes donor and acceptor moieties). The manufacture of these duallabeled probes generally involves synthesis, purification, and qualitycontrol processes that are complex, labor intensive, and expensive. Inaddition, the baseline fluorescence of dual labeled probes typicallymust fall within a specified range for optimum performance. Further,certain dual labeled probes may suffer from instability that results inbaseline drift, which negatively impacts shelf-life. Moreover, theinsertion of an internal label typically leads to duplex destabilizationupon hybridization, which must generally be compensated for.

All of these problems can be circumvented if unquenched single-labeledprobes are used for detecting the products of nucleic acid amplificationreactions. For example, the use of ethidium bromide and several otherDNA binding dyes to quench the fluorescence of oligonucleotides in alength dependent manner has been described. However, these dyesgenerally cannot be used for real time detection, e.g., due to their lowDNA binding affinity at higher temperatures. Accordingly, there exists aneed for nucleic acid amplification reaction mixture additives that havethe ability to bind and quench single-labeled probes at highertemperatures typically utilized for real time detection.

In addition, multiplex nucleic acid amplification detection using 5′nuclease probes, molecular beacons, or FRET probes, among otherdetection methods, typically includes the pooling of quenched orunquenched fluorescent probes, e.g., to improve assay throughputrelative to protocols that utilize single probes in a given reaction. Toillustrate, multiplex assays are commonly used to detect multiplegenotype markers or pathogens in samples obtained from patients as partof diagnostic procedures. In these formats, the overall baseline orbackground fluorescence from the pooled probes increases additively asthe number of probes increases in the reaction mixture. This baselinefluorescence also increases in essentially any assay system when theamount of a single probe is increased. Baseline fluorescence generallyadversely affects the performance of a given assay by, for example,reducing the detection sensitivity and dynamic range of the assay.Accordingly, baseline fluorescence effectively limits the total numberof fluorescent probes and/or the amount of a given probe that can beadded to a particular assay.

Although a wide variety of DNA hybridization and amplificationstrategies are known in the art, certain challenges remain. For example,the high levels of sequence divergence (i.e., sequence heterogeneity) inRNA/DNA viruses such as HIV, HCV and HPV make it particularly difficultto standardize methods for nucleic acid amplification, genotyping and/ordetection. This viral sequence heterogeneity prevents the development ofassays that have uniformly high sensitivity for all different viralgenotypes and subtypes. Sequence differences between the experimentaltarget and the primers and/or probes (e.g., probes for viral detectionand/or genotyping) that result in duplex mismatches compromise assayperformance, and can result in false negative results ormisclassification. Failure to detect the multitude of relevant viralgenotypes can have significant negative consequences, particularly inapplications such as screening of clinical samples.

Quantitative assays (e.g., assays for assessing viral load) are evenmore vulnerable to sequence heterogeneity of the analytes, as the loweramplification/detection efficiencies might be falsely attributed tolower amounts of target present in a sample (in the absence ofdefinitive genotype information). Because nucleic acid-based assaysdepend on hybridization, primer/probe mismatches can significantlyreduce the accuracy of the quantitation.

In order to minimize these differences, primers and probes arepreferably selected from conserved regions of viral genomes. However,this is becoming increasingly difficult in view of two primary factors,(i) many viruses, e.g., HIV and influenza, display rapid rates ofmutagenesis and genome evolution, and (ii) the number of known viralgenotypes and subtypes continues to grow, where the newly discoveredisolates continue to expand the scope of known genomic diversity. Insome cases, assigning viral genotype information is critical for patientstratification and therapy decisions, as differences are observed in theresponse to therapy based on the viral genotype. In these cases, it ismore desirable to amplify and detect relatively less conserved regionsof the viral genome in order to adequately differentiate between thevarious genotypes.

Primer/probe mismatches can be overcome to a limited extent by includinga multiplicity of genotype specific primers and probes, oralternatively, by incorporating base analogs that increase the stabilityof DNA-DNA or RNA-RNA duplexes. However, these solutions are of limitedutility and result in vastly increased assay complexity and cost.Although sequencing provides the highest resolution in genotypeassignment, its application in a high-throughput clinical settingremains unfeasible.

As illustrated above, there is a need in the art for improved methodsfor nucleic acid analysis. For example, there is a need in the art forimproved methods for nucleic acid detection, identification,amplification, characterization (e.g., Tm determination) andquantitation, especially where sequence heterogeneity and duplexmismatches can interfere with currently used methods. In the discussionabove, the challenges of nucleic acid analysis are illustrated in thecontext of amplification, detection and genotyping of viral targets.However, these challenges are not unique to viral targets, and indeed,find relevance to a wide variety of nucleic acid analysis applications,such as microbial pathogen testing, genetic testing, and environmentaltesting.

SUMMARY OF THE INVENTION

The present invention provides methods of modulating the emission oflight (e.g., baseline light emissions) from labeled nucleic acids,including 5′-nuclease probes. For example, certain light emissionmodifiers described herein reduce light emissions from labeled probes insolution at elevated temperatures and under other reaction conditionstypically used for real-time detection. Moreover, unlike various otherpreviously known solution quenchers, the light emission modifiers of theinvention are not detrimental to the performance of nucleic acidamplification reactions and retain sufficient nucleic acid bindingaffinity at the elevated temperatures commonly utilized in thesereactions such that real-time detection can be effected. The approachesto real-time detection described herein include the use ofsingle-labeled probes, multi-labeled probes, or both types of probestogether in a given reaction mixture. In addition to reaction mixturesand methods of modifying light emissions from labeled probes, relatedkits and systems are also provided.

In one aspect, the invention provides a reaction mixture that includesat least one labeled oligonucleotide. The oligonucleotide (e.g., asingle-stranded oligonucleotide, etc.) is labeled with at least onelight-emitting moiety (e.g., a fluorescent dye or the like). Thereaction mixture also includes at least one soluble light emissionmodifier that modifies (e.g., reduces, etc.) a light emission from thelabeled oligonucleotide. In some embodiments, the labeledoligonucleotide comprises a 5′-nuclease probe.

In another aspect, the invention provides a reaction mixture thatincludes at least one oligonucleotide that comprises at least twolabeling moieties in which at least one of the labeling moieties islight-emitting. In some embodiments, for example, the oligonucleotidecomprises a 5′-nuclease probe. The reaction mixture also includes atleast one light emission modifier (e.g., a soluble light emissionmodifier, etc.) that modifies a baseline emission of light from theoligonucleotide at a temperature of at least about 40° C. In certainembodiments, the light emission modifier reduces the baseline emissionof light from the oligonucleotide.

In another aspect, the invention relates to a reaction mixture thatincludes at least one oligonucleotide (e.g., a 5′-nuclease probe, etc.)that comprises at least two labeling moieties in which at least one ofthe labeling moieties is light-emitting. This reaction mixture alsoincludes at least one diazine dye and/or thiazine dye that reduces abaseline emission of light from the oligonucleotide. Typically, thediazine dye and/or thiazine dye reduces the baseline emission of lightfrom the oligonucleotide at a temperature of at least about 40° C.

In some embodiments, the reaction mixtures described herein comprise aplurality of oligonucleotides that are used, e.g., as part of amultiplexed 5′-nuclease reaction or other application. In theseembodiments, at least one of the oligonucleotides generally comprises atleast one labeling moiety that differs from a labeling moiety of anotheroligonucleotide. Typically, the different labeling moieties comprisedifferent light-emitting labeling moieties (e.g., different fluorescentdyes, etc.) and the light emission modifier (e.g., a diazine dye, athiazine dye, and/or the like) modifies (e.g., reduces) baselineemissions of light from each of the oligonucleotides. In someembodiments, the reaction mixtures of the invention are packaged inkits.

The reaction mixtures described herein optionally include various othercomponents. In some embodiments, for example, reaction mixtures includecomponents that are useful in performing nucleic acidamplification/detection assays, such as one or more of: a buffer, asalt, a metal ion, a nucleotide incorporating biocatalyst having a 5′ to3′ nuclease activity (e.g., a Taq DNA polymerase, etc.), apyrophosphatase, a primer nucleic acid, a template nucleic acid, anamplicon, a nucleotide, glycerol, dimethyl sulfoxide, poly rA (oranother carrier nucleic acid), or the like. The reaction mixtures andother related aspects of the invention typically substantially lackethidium bromide.

In one aspect, the invention provides a method of detecting a targetnucleic acid in a sample. The method includes (a) providing at least onelabeled oligonucleotide (e.g., a 5′-nuclease probe, etc.). Theoligonucleotide is labeled with at least one light emitting moiety. Inaddition, at least a subsequence of the labeled oligonucleotide issufficiently complementary to at least a subsequence of at least onetarget nucleic acid and/or to at least a subsequence of an amplicon ofthe target nucleic acid such that the labeled oligonucleotide hybridizeswith the target nucleic acid and/or the amplicon of the target nucleicacid under at least one selected condition (e.g., an annealingtemperature, an extension temperature, and/or the like). The method alsoincludes (b) providing at least one soluble light emission modifier thatmodifies a light emission from the labeled oligonucleotide to a greaterextent than from a labeled fragment of the oligonucleotide. In addition,the method includes (c) amplifying the nucleic acid in the sample in thepresence of the labeled oligonucleotide and the soluble light emissionmodifier in an amplification reaction that comprises the selectedcondition such that the labeled oligonucleotide, hybridized with thetarget nucleic acid or the amplicon of the target nucleic acid, iscleaved to produce at least one labeled oligonucleotide fragment. Themethod further includes (d) detecting light emission at least from thelabeled oligonucleotide fragment during (c), e.g., as a part of areal-time monitoring process.

In another aspect, the invention provides a method of modifying abaseline emission of light from a labeled oligonucleotide. The methodincludes (a) providing at least one oligonucleotide that comprises atleast two labeling moieties in which at least one of the labelingmoieties is light-emitting. The method also includes (b) contacting theoligonucleotide with at least one light emission modifier (e.g., adiazine dye, a thiazine dye, and the like) that modifies a baselineemission of light from the oligonucleotide at a temperature of at leastabout 40° C. (e.g., under conditions of real-time detection, etc.). Incertain embodiments, (b) comprises contacting the oligonucleotide andthe light emission modifier in solution. Typically, the method alsoincludes detecting light emission from the labeled oligonucleotidebefore, during, and/or after (b).

In still another aspect, the invention provides a method of reducing abaseline emission of light from a labeled oligonucleotide. The methodincludes (a) providing at least one oligonucleotide that comprises atleast two labeling moieties in which at least one of the labelingmoieties is light-emitting. The method further includes (b) contactingthe oligonucleotide with at least one diazine dye and/or thiazine dye,thereby reducing the baseline emission of light from the labeledoligonucleotide. In some embodiments, the oligonucleotide and thediazine dye and/or thiazine dye are contacted at a temperature of atleast about 40° C. Typically, (b) includes contacting theoligonucleotide and the diazine dye and/or thiazine dye in solution.Moreover, the method generally includes detecting light emission fromthe labeled oligonucleotide before, during, and/or after (b).

In certain embodiments of the methods described herein, the methodscomprise amplifying at least one target nucleic acid. Typically, atleast a subsequence of the oligonucleotide is sufficiently complementaryto at least a subsequence of the target nucleic acid and/or to at leasta subsequence of an amplicon of the target nucleic acid such that theoligonucleotide hybridizes with the target nucleic acid and/or theamplicon of the target nucleic acid. In some of the embodiments, forexample, the oligonucleotide comprises a 5′-nuclease probe and themethod comprises amplifying the target nucleic acid under conditionswhereby the 5′-nuclease probe is cleaved. In these embodiments, themethod generally includes detecting cleavage of the 5′-nuclease probe.The target nucleic acid typically correlates with a diagnosis of atleast one genetic disorder and/or at least one disease state for asubject that comprises a copy of the target nucleic acid.

In some multiplexing embodiments of the methods described herein, themethods include contacting a plurality of oligonucleotides with thelight emission modifier (e.g., a diazine dye, a thiazine dye, and/or thelike). In these embodiments, at least one of the oligonucleotidestypically comprises at least one labeling moiety that differs from alabeling moiety of another oligonucleotide. The different labelingmoieties generally comprise different light-emitting labeling moietiesand the light emission modifier modifies (e.g., reduces) baselineemissions of light from each of the oligonucleotides.

To further illustrate, some embodiments of the methods described hereincomprise contacting one or more single-labeled oligonucleotides (e.g., a5′-nuclease probe, etc.) with the light emission modifier (e.g., adiazine dye, a thiazine dye, and/or the like). Typically, a labelingmoiety of at least one of the single-labeled oligonucleotides islight-emitting and the light emission modifier modifies (e.g., reduces)an emission of light from the light-emitting single-labeledoligonucleotide.

In another aspect, the invention provides a kit that includes (a) atleast one light emission modifier (e.g., one or more dyes selected froma diazine dye, a thiazine dye, and the like) that modifies baselineemissions of light from labeled oligonucleotides at a temperature of atleast about 40° C. The kit also includes (b) instructions for modifyinga light emission (e.g., a baseline emission of light, etc.) from atleast one oligonucleotide that comprises at least one light-emittinglabeling moiety with the light emission modifier. Generally, the kitincludes at least one container for packaging the light emissionmodifier and/or the instructions.

In still another aspect, the invention provides a kit that includes (a)at least one diazine dye and/or thiazine dye. The kit also includes (b)instructions for reducing a light emission from at least oneoligonucleotide that comprises at least one light-emitting labelingmoiety with the diazine dye and/or thiazine dye. The kit also typicallyincludes at least one container for packaging the diazine dye and/orthiazine dye and/or the instructions.

In some embodiments, the kits described herein also include variousother components. To illustrate, these kits optionally include at leastone primer nucleic acid that is at least partially complementary to atleast one subsequence of a target nucleic acid. In certain embodiments,the kits include the oligonucleotide. Optionally, the oligonucleotidecomprises a 5′-nuclease probe. In some embodiments, the kits include atleast one single-labeled oligonucleotide that comprises a light-emittinglabeling moiety. For example, the single-labeled oligonucleotideoptionally comprises a primer nucleic acid that is at least partiallycomplementary to at least one subsequence of at least one target nucleicacid. In embodiments of these kits that include primer nucleic acids,the kits also typically include instructions for amplifying one or moresegments of the target nucleic acid with the primer nucleic acid, atleast one nucleotide incorporating biocatalyst having a 5′ to 3′nuclease activity, and one or more nucleotides. In these embodiments,the kits also generally include at least one nucleotide incorporatingbiocatalyst having a 5′ to 3′ nuclease activity and/or one or morenucleotides.

In another aspect, the invention relates to a system that includes (a)at least one oligonucleotide (e.g., a 5′-nuclease probe, etc.) thatcomprises at least one light-emitting labeling moiety. The system alsoincludes (b) at least one light emission modifier (e.g., at least onediazine dye, thiazine dye, and/or the like) that modifies a baselineemission of light from the oligonucleotide at a temperature of at leastabout 40° C. In addition, the system also includes (c) at least onedetector that detects light emitted from the oligonucleotide and/or atleast one fragment of the oligonucleotide.

In yet another aspect, the invention provides a system that includes (a)at least one oligonucleotide (e.g., a 5′-nuclease probe, etc.) thatcomprises at least one light-emitting labeling moiety. In addition, thesystem also includes (b) at least one diazine dye and/or thiazine dye,and (c) at least one detector that detects light emitted from theoligonucleotide and/or at least one fragment of the oligonucleotide.

In certain embodiments, the systems described herein include certainother components. For example, the systems optionally include at leastone logic device operably connected to the detector. The logic devicegenerally includes one or more instruction sets that scale detectedlight emissions relative to one another. In some embodiments of thesesystems, at least one container comprises the oligonucleotide and thelight emission modifier (e.g., at least one diazine dye, thiazine dye,and/or the like). In these embodiments, the systems typically include(d) at least one thermal modulator that thermally communicates with thecontainer to modulate temperature in the container, and/or (e) at leastone fluid transfer component that transfers fluid to and/or from thecontainer. Generally, the oligonucleotide and the light-emittinglabeling moiety are present in solution. In some embodiments, thecontainer also includes components that can be used to perform variousnucleic acid amplification-based assays, such as one or more of, e.g., abuffer, a salt, a metal ion, a nucleotide incorporating biocatalysthaving a 5′ to 3′ nuclease activity, a pyrophosphatase, a primer nucleicacid, a template nucleic acid, an amplicon, a nucleotide, glycerol,dimethyl sulfoxide, poly rA, or the like.

The light emission modifiers utilized in the reaction mixtures, methods,kits, and systems described herein generally include soluble quenchermoieties. In some embodiments, light emission modifiers substantiallylack intrinsic fluorescence at least under selected light emissiondetection conditions (e.g., at detection wavelengths of 600 nm or less,etc.). Typically, the light emission modifiers described hereinassociate (e.g., intercalate, bind to, or the like) witholigonucleotides, such as the 5′-nuclease probes described herein. Incertain embodiments, for example, a light emission modifier comprisesone or more dyes selected from, e.g., a diazine dye, a thiazine dye, andthe like. Exemplary diazine dyes include an azocarmine dye, a phenazinedye, diethylsafraninazodimethylaniline chloride (i.e., Janus Green B),and the like. Examples of suitable thiazine dyes include methylene blue,methylene green, thionin, 1,9-dimethylmethylene blue,sym-dimethylthionin, toluidine blue O, new methylene blue, methyleneviolet bernthsen, azure A, azure B, azure C, and the like.

The invention provides a variety of compositions and methods that finduse in the detection, amplification and analysis of nucleic acids. Morespecifically, these methods utilize advantageous and previouslyunidentified properties of thiazine and diazine dyes.

In some embodiments, the methods of the invention take advantage of thepreviously unidentified ability of thiazine dyes to stabilize nucleicacid duplexes. This method is broadly applicable to any nucleic acidmanipulation that uses duplex nucleic acid molecules and/orhybridization methodologies. Essentially, the methods for producing astabilized nucleic acid duplex use the steps of (a) providing a samplecontaining or suspected of containing a target nucleic acid molecule; anoligonucleotide complementary or partially complementary to the targetnucleic acid molecule; and at least one thiazine dye present at aconcentration effective to stabilize a duplex formed between the targetnucleic acid molecule and the oligonucleotide; and (b) alternatively (i)annealing the target nucleic acid and the oligonucleotide in thepresence of the thiazine dye; or (ii) annealing the target nucleic acidand the oligonucleotide, followed by admixing with the thiazine dye;under conditions where a duplex can form to produce at least onestabilized nucleic acid duplex. In these methods, stability of thenucleic acid duplex of (b) is improved compared to the stability of thesame nucleic acid duplex comprising the target nucleic acid and theoligonucleotide in the absence of the thiazine dye or a reducedconcentration of the thiazine dye. These methods optionally include thedemonstration of stability of the nucleic acid duplexes in the presenceand absence or reduced concentration of the thiazine dye, which can beaccomplished by any suitable method, for example, including (i) amelting temperature (T_(m) analysis); (ii) a C_(T) determination; or(iii) a 5′-nuclease assay. In some aspects, the methods optionallyinclude detecting the stabilized nucleic acid duplex under theconditions that provide improved stability.

It is not intended that the type, nature, configuration, structure orsequence of the duplex that is stabilized be limited in any respect. Forexample, in these methods, the stabilized nucleic acid duplex cancomprise one or more, two or more, or three or more nucleobasemismatches. Perfect match duplexes can also be stabilized. In someaspects, the oligonucleotide in the stabilized duplex is effective toprime a nucleic acid extension reaction when annealed to the targetnucleic acid. In some embodiments, the hybridization reactions are partof a PCR amplification reactions, where a pair of oligonucleotides areused in the hybridization with the target nucleic acid molecule, whereeach of the oligonucleotide primers is effective to prime a nucleic acidextension reaction when annealed to the target nucleic acid. In someaspects, the hybridization reaction includes a labeled oligonucleotideprobe that is complementary or partially complementary to the targetnucleic acid molecule. In some aspects, the target nucleic acid moleculeis an amplicon.

The nucleic acids used in the methods of the invention are not limitedto naturally occurring oligomeric structures or naturally occurringbases. For example, one or more of the molecules in the duplex cancomprises one or more naturally-occurring nucleotides, modifiednucleotides, nucleotide analogs, one or more unnatural bases, unnaturalinternucleotide linkages, unnatural nucleotide backbones, or anycombination thereof.

Typically, in the methods for stabilizing nucleic acid duplexes, thestabilized hybridization complex is an intermolecular hybridizationcomplex, where the antiparallel hybridizing strands are two separatenucleic acid molecules. However, in some adaptations of the methods forstabilizing nucleic acid duplexes, the stabilized hybridization complexis an intramolecular hybridization complex, where the antiparallelhybridizing strands are actually on a single nucleic acid molecule, suchas in the case of a molecular beacon type configuration.

A requirement for these methods for duplex stabilization is the presenceof a thiazine dye. Any thiazine dye can be used, for example but notlimited to, methylene blue, methylene green, thionin,sym-dimethylthionin, toluidine blue O, new methylene blue, methyleneviolet bernthsen, azure A, azure B, azure C and 1,9-dimethylmethyleneblue. The concentration of the dye that is used to improve the stabilityof the duplex is not particularly limited. In some aspects, at least onethiazine dye is present at a concentration of at least 10 μg/mL. In someaspects, the annealing comprises annealing in the presence of a thiazinedye at a concentration between about 10 μg/mL and 50 μg/mL, oralternatively, at a concentration between about 20 μg/mL and 40 μg/mL.In some embodiments, the thiazine dye is sued at a concentration ofabout 40 μg/mL.

In some embodiments, the invention provides kits for the execution ofthe methods for stabilizing nucleic acid duplexes. These kits cancontain any reagents or other components that are required for orsimplify any of the methods for duplex stabilization. In some aspects,these kits can contain an oligonucleotide complementary or partiallycomplementary to a target nucleic acid molecule of interest; and atleast one thiazine dye present at a concentration effective to stabilizea duplex formed between the target nucleic acid molecule and theoligonucleotide. The kits of the invention can include instructions tothe kit user, and can also include one or more containers for holdingall or any subset of components of the kit.

In some aspects, the invention provides integrated systems for theexecution of the methods for stabilizing nucleic acid duplexes. Thesystems can include instrumentation and means for interpreting andanalyzing collected data, especially where the collected data is subjectto subsequent analysis using algorithms and/or electronically storedinformation (e.g., analysis of collected fluorescence data, etc). Eachpart of an integrated system is functionally interconnected, and in somecases, physically connected. In some embodiments, the integrated systemis automated, where there is no requirement for any manipulation of thesample or instrumentation by an operator following initiation of themethods. A system of the invention can include instrumentation. Forexample, the invention can include a detector such as a fluorescencedetector (e.g., a fluorescence spectrophotometer), and a thermal cyclingdevice, or thermocycler. In some embodiments, the thermal cycling deviceand the detector are an integrated instrument, where the thermal cyclingand emission detection (e.g., fluorescence detection) are done in thesame device. A detector, e.g., a fluorescence spectrophotometer, can beconnected to a computer for controlling the spectrophotometeroperational parameters and/or for storage of data collected from thedetector. The computer may also be operably connected to the thermalcycling device to control the temperature, timing, and/or rate oftemperature change in the system. The integrated computer can alsocontain the “correlation module” where the data collected from thedetector is analyzed. In some embodiments, the correlation modulecomprises a computer program that calculates.

A variety of uses of the thiazine dyes and diazine dyes as soluble lightemission modifiers are provided herein. In one aspect, the methods ofthe invention take advantage of the previously unidentified lightemission modifying properties of thiazine and diazine dyes by employingthe dyes as soluble quenchers in a donor/quencher pair. In traditionalFRET configurations, the FRET quencher moiety is typically integratedinto the same nucleic acid molecule as the FRET donor, or alternatively,is integrated into a separate nucleic acid molecule. The inventionprovides methods that are a simplification over the methods used in theart, where the invention provides methods where the quencher moiety isreplaced by a soluble quencher molecule that can be a thiazine dye or adiazine dye, or any molecule that is structurally related thereto thatretains the required light-quenching property. The range of uses of thethiazine dyes and diazine dyes as soluble quenchers is not limited, andindeed, can be adapted for use in most instances where a traditionalquenching moiety is used.

For example, in some aspects, the invention provides methods fordetermining the melting temperature (T_(m)) of a hybridization complex,the method comprising the steps:

(a) providing, (i) a probe comprising a light emitting moiety; (ii) ahybridization target that is complementary or partially complementary tothe probe; and (iii) a soluble light emission modifier comprising athiazine dye or a diazine dye, where the soluble light emission modifieris capable of quenching the light emitting moiety;(b) annealing the probe with the hybridization target under conditionswhere base-pairing can occur to form a target hybridization complex;(c) altering the temperature of the target hybridization complex in thepresence of the soluble light emission modifier and measuring anemission of the light emitting moiety;(d) correlating the measured emission of the light emitting moiety withthe presence of the target hybridization complex as a function oftemperature, thereby determining Tm of the target hybridization complexbased on the measured emission.

In these Tm determination methods, the light emitting moiety can be adonor moiety, and the light emission modifier can be a quencher. Inthese methods, altering the temperature can be raising the temperature(melting curve) or lowering the temperature (annealing curve). In someaspects, a range of temperatures is used in the measuring step, forexample, a range of temperatures of about 20° C. to about 95° C.

In some aspects, the hybridization target is an amplicon correspondingto a nucleic acid target, where the amplicon is typically generated by apolymerase chain reaction (e.g., in an asymmetric PCR amplification). Inmost instances where PCR is used, the PCR amplification uses anamplification primer pair specific for a target nucleic acid ofinterest, a thermostable DNA-dependent DNA polymerase, freedeoxyribonucleotide triphosphates and a suitable DNA polymerase reactionbuffer. In some embodiments, the amplicon generation is by reversetranscribing an RNA nucleic acid target and amplifying by a polymerasechain reaction (RT-PCR).

The targets for the Tm analysis are not limited in any aspect. In someembodiments, the nucleic acid target is a viral genome. The nucleic acidtarget can optionally be provided in a sample, which can be, forexample, human blood, serum or plasma.

The methods for Tm determination contain at least one soluble quencherwhich can be a diazine dye or a thiazin dye; for example but not limitedto, methylene blue, methylene green, thionin, sym-dimethylthionin,toluidine blue O, new methylene blue, methylene violet bernthsen, azureA, azure B, azure C, 1,9-dimethylmethylene blue, azocarmine dye, aphenazine dye, and diethylsafraninazodimethylaniline chloride.

The nucleic acids used in the methods of the invention for Tmdetermination are not limited to naturally occurring oligomericstructures or naturally occurring bases. For example, one or more of themolecules in the duplex can comprises one or more naturally-occurringnucleotides, modified nucleotides, nucleotide analogs, one or moreunnatural bases, unnatural internucleotide linkages, unnaturalnucleotide backbones, or any combination thereof.

In some embodiments, the invention provides kits for the execution ofthe methods for Tm determination. These kits can contain any reagentsthat are required for or simplify use of the methods for Tmdetermination. In some aspects, these methods can contain (a) at leastone probe comprising a light emitting moiety, where the probe iscomplementary or partially complementary to a hybridization target ofinterest; (b) at least one soluble light emission modifier comprising athiazine dye or a diazine dye, where the soluble light emission modifieris capable of quenching the light emitting moiety; and (c) one or morecontainers comprising the probe, the soluble light emission modifier, orboth the probe and light emission modifier. In some aspects, the lightemitting moiety is a FRET donor moiety. In some aspects, the lightemission modifier is a FRET quencher. In some embodiments, the kits alsocontain instructions for determining the T_(m) of a hybridizationcomplex comprising the probe and the hybridization target.

Optionally, the kits of the invention for determining Tm can include oneor more additional components selected from a reverse transcriptase, atleast one primer suitable for reverse transcriptase initiation from anRNA target, a thermostable DNA-dependent DNA polymerase and/or a enzymehaving both DNA-dependent and RNA-dependent (i.e., reversetranscriptase) polymerase activities, free deoxyribonucleotidetriphosphates, standardization samples, positive control samples,negative control samples, buffers suitable for enzymatic reactions,sample collection tubes and amplification reaction tubes.

In some aspects, the invention provides integrated systems for theexecution of the methods for Tm determination. The systems can includeinstrumentation and means for interpreting and analyzing collected data,especially where the collected data is subject to subsequent analysisusing algorithms and/or electronically stored information (e.g.,collected fluorescence data is translated into a Tm value). Each part ofan integrated system is functionally interconnected, and in some cases,physically connected. The systems of the invention for conducting Tmdetermination of a hybridization complex include:

(a) a sample or reaction mixture comprising (i) a nucleic acid probecomprising a light emitting moiety that emits a signal; (ii) a targetnucleic acid that is complementary or partially complementary to thenucleic acid probe; and (iii) a thiazine dye or a diazine dye;(b) a thermal control device for regulating the temperature of thesample or reaction mixture over a range of temperatures, where the rangeincludes: (i) a temperature where essentially all probe molecules annealwith the hybridization target at a given set of hybridizationconditions; (ii) a temperature where 50% of the target hybridizationcomplexes are dissociated at the hybridization conditions, and (iii) atemperature where essentially no probe molecules anneal with thehybridization target and essentially no hybridization complexes arepresent at the hybridization conditions;(c) a detector for measuring the signal from the sample over the rangeof temperatures; and(d) a correlation module that is operably coupled to the detector andreceives signal measurements over the range of temperatures, where thecorrelation module correlates the signal intensity with the presence ofa hybridization complex comprising the probe and the hybridizationtarget in admixture with the thiazine dye or diazine dye as a functionof temperature, thereby determining the T_(m) of the targethybridization complex.

In some aspects, the light emitting moiety is a FRET donor moiety.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularoligonucleotides, methods, compositions, kits, systems, computers, orcomputer readable media, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. Further, unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. In describing and claiming the present invention, thefollowing terminology and grammatical variants will be used inaccordance with the definitions set forth below.

The term “5′ to 3′ nuclease activity” refers to a 5′ to 3′ exonucleaseactivity associated with some nucleotide incorporating biocatalysts,such as nucleic acid polymerases in which nucleotides are sequentiallyremoved from the 5′ end of an oligonucleotide, a 5′ to 3′ endonucleaseactivity in which cleavage occurs more than one phosphodiester bond(nucleotide) from the 5′ end, or both activities. An exemplary substratefor 5′ to 3′ endonuclease activity-dependent cleavage on aprobe-template hybridization complex is a displaced single-strandednucleic acid, a fork-like structure, with hydrolysis occurring at thephosphodiester bond joining the displaced region with the base-pairedportion of the strand, as discussed in, e.g., Holland et al. (1991)Proc. Natl. Acad. Sci. USA 88:7276-80, which is incorporated byreference.

A “5′-nuclease probe” refers to an oligonucleotide that comprises atleast one light emitting labeling moiety and that is used in a5′-nuclease reaction to effect target nucleic acid detection. In someembodiments, for example, a 5′-nuclease probe includes only a singlelight-emitting moiety (e.g., a fluorescent dye, etc.). In certainembodiments, 5′-nuclease probes include regions of self-complementaritysuch that the probes are capable of forming hairpin structures underselected conditions. Typically, the light emission modifiers describedherein modify light emission from intact, full-length 5′-nuclease probesto a greater extent than from labeled fragments of such probes, whichfragments are generated from the full-length probes during exo- and/orendonucleolytic cleavage steps of 5′-nuclease reactions. To furtherillustrate, in some embodiments a 5′-nuclease probe comprises at leasttwo labeling moieties and emits radiation of increased intensity afterone of the two labels is cleaved or otherwise separated from theoligonucleotide. In certain embodiments, for example, a 5′-nucleaseprobe is labeled with two different fluorescent dyes, e.g., a 5′terminus reporter dye and the 3′ terminus quencher dye or moiety. Insome embodiments, 5′-nuclease probes are labeled at one or morepositions other than, or in addition to, terminal positions. When theprobe is intact, energy transfer typically occurs between the twofluorophores such that fluorescent emission from the reporter dye isquenched at least in part. During an extension step of a polymerasechain reaction, for example, a 5′-nuclease probe bound to a templatenucleic acid is cleaved by the 5′ to 3′ nuclease activity of, e.g., aTaq polymerase or another polymerase having this activity such that thefluorescent emission of the reporter dye is no longer quenched.Exemplary 5′-nuclease probes are also described in, e.g., U.S. Pat. No.5,210,015, entitled “HOMOGENEOUS ASSAY SYSTEM USING THE NUCLEASEACTIVITY OF A NUCLEIC ACID POLYMERASE,” issued May 11, 1993 to Gelfandet al., U.S. Pat. No. 5,994,056, entitled “HOMOGENEOUS METHODS FORNUCLEIC ACID AMPLIFICATION AND DETECTION,” issued Nov. 30, 1999 toHiguchi, and U.S. Pat. No. 6,171,785, entitled “METHODS AND DEVICES FORHEMOGENEOUS NUCLEIC ACID AMPLIFICATION AND DETECTOR,” issued Jan. 9,2001 to Higuchi, which are each incorporated by reference. In otherembodiments, two different probes are used, one labeled with a reporterdye and the other with a quencher dye, in an arrangement such thatfluorescent resonance energy transfer can occur when both are hybridizedto the target nucleic acid. In still other embodiments, a 5′ nucleaseprobe may be labeled with two or more different reporter dyes and the 3′terminus quencher dye or moiety.

A “5′ nuclease reaction” or “5′ nuclease assay” of target or template,primer, and probe (e.g., 5′-nuclease probes, etc.) nucleic acids refersto the degradation of a probe hybridized to the template nucleic acidwhen the primer is extended by a nucleotide incorporating biocatalysthaving 5′ to 3′ nuclease activity, as described further below. 5′nuclease reactions are also described in, e.g., U.S. Pat. No. 6,214,979,entitled “HOMOGENEOUS ASSAY SYSTEM,” issued Apr. 10, 2001 to Gelfand etal., U.S. Pat. No. 5,804,375, entitled “REACTION MIXTURES FOR DETECTIONOF TARGET NUCLEIC ACIDS,” issued Sep. 8, 1998 to Gelfand et al., U.S.Pat. No. 5,487,972, entitled “NUCLEIC ACID DETECTION BY THE 5′-3′EXONUCLEASE ACTIVITY OF POLYMERASES ACTING ON ADJACENTLY HYBRIDIZEDOLIGONUCLEOTIDES,” issued Jan. 30, 1996 to Gelfand et al., and U.S. Pat.No. 5,210,015, supra, which are each incorporated by reference.

An “amplicon” refers to a molecule made by amplifying a nucleic acidmolecule, e.g., as occurs in a nucleic acid amplification reaction, suchas a polymerase chain reaction (“PCR”), a strand displacementamplification (SDA), transcription mediated amplification (TMA), ligasechain reaction (LCR), or other nucleic acid amplification technique.Typically, an amplicon is a copy of a selected nucleic acid (e.g., atemplate or target nucleic acid) or is complementary thereto.

An “amplification reaction” refers to a reaction involving thereplication of one or more target nucleic acid sequences or complementsthereto. Exemplary amplification reactions include PCR, ligase chainreactions (LCR), among many others.

The term “baseline emission of light” in the context of a labeledoligonucleotide refers a detectable emission of light from theoligonucleotide prior to being contacted with a light emission modifier.Certain 5′-nuclease probes, for example, emit detectable amounts ofresidual light despite the presence of one or more quencher moietiesincorporated into the probe design. This baseline or background lightemission tends to limit the signal to noise ratio of 5′-nucleasereactions. Moreover, this baseline emission of light generally increasesadditively in multiplexed detection formats where multiple labeledprobes are pooled with one another. This additive increase in thebaseline emission of light also occurs when the amount of a single probeis increased in a given application.

The term “cleavage” in the context of 5′-nuclease reactions refers tothe degradation or fragmentation (hydrolysis) of 5′-nuclease probes bythe 5′ to 3′ nuclease activity associated with various polymerasestypically utilized in those reactions.

A “complement” of a nucleic acid refers to at least a nucleic acidsegment that can combine in an antiparallel association or hybridizewith at least a subsequence of that nucleic acid. The antiparallelassociation can be intramolecular, e.g., in the form of a hairpin loopwithin a nucleic acid, or intermolecular, such as when two or moresingle-stranded nucleic acids hydridize with one another. Certain basesnot commonly found in natural nucleic acids may be included in thenucleic acids referred to herein and include, for example, inosine,7-deazaguanine and those discussed below. Complementarity need not beperfect; stable duplexes, for example, may contain mismatched base pairsor unmatched bases. Those skilled in the art of nucleic acid technologycan determine duplex stability by empirically considering a number ofvariables including, for example, the length of a region ofcomplementarity, base composition and sequence of nucleotides in aregion of complementarity, ionic strength, and incidence of mismatchedbase pairs.

“Corresponding” means identical to or complementary to a designatedsequence of nucleotides in a nucleic acid. The exact application of theterm will be evident to one of skill in the art by the context in whichthe term is used.

A “diazine dye” refers to any of a class of organic chemical compoundscontaining a benzene ring in which two of the carbon atoms have beenreplaced by nitrogen atoms. Exemplary diazine dyes include an azocarminedye, a phenazine dye, and diethylsafraninazodimethylaniline chloride(Janus Green B or Diazine Green 5).

Nucleic acids “hybridize” when complementary single strands of nucleicacid pair to give a double-stranded nucleic acid sequence. Hybridizationoccurs due to a variety of well-characterized forces, including hydrogenbonding, solvent exclusion, and base stacking. An extensive guide tonucleic hybridization may be found in Tijssen, Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” Elsevier (1993).

The phrase “in solution” refers to an assay or reaction condition inwhich the components of the assay or reaction are not attached to asolid support in a fluid medium.

A “label” or “labeling moiety” refers to a moiety attached (covalentlyor non-covalently), or capable of being attached, to a molecule, whichmoiety provides or is capable of providing information about themolecule (e.g., descriptive, identifying, etc. information about themolecule) or another molecule with which the labeled molecule interacts(e.g., hybridizes, etc.). Exemplary labels include fluorescent labels(including, e.g., quenichers or absorbers), non-fluorescent labels,colorimetric labels, chemiluminescent labels, bioluminescent labels,radioactive labels, mass-modifying groups, antibodies, antigens, biotin,haptens, enzymes (including, e.g., peroxidase, phosphatase, etc.), andthe like. To further illustrate, fluorescent labels may include dyesthat are negatively charged, such as dyes of the fluorescein family, ordyes that are neutral in charge, such as dyes of the rhodamine family,or dyes that are positively charged, such as dyes of the cyanine family.Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NANand ZOE. Dyes of the rhodamine family include, e.g., Texas Red, ROX,R110, R6G, and TAMRA. FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, andTAMRA are commercially available from, e.g., Perkin-Elmer, Inc.(Wellesley, Mass., USA), and Texas Red is commercially available from,e.g., Molecular Probes, Inc. (Eugene, Oreg.). Dyes of the cyanine familyinclude, e.g., Cy2, Cy3, Cy5, and Cy7, and are commercially availablefrom, e.g., Amersham Biosciences Corp. (Piscataway, N.J., USA).Additional labels are referred to herein or are otherwise known in theart.

A “light emission modifier” refers to a substance that non-covalentlyassociates with a nucleic acid in a mixture and that changes thedetectable emission of radiation from a radiation source associated withthe nucleic acid when the substance is proximal to the radiation source.In some embodiments, for example, certain light emission modifiersdescribed herein reduce or quench the emission of light that wouldotherwise be emitted (e.g., a baseline emission of light) fromoligonucleotides that include at least one light-emitting moiety (e.g.,5′-nuclease probes, etc.) when the light emission modifiers arecontacted with those oligonucleotides. Light emission modifiers aretypically soluble and in these embodiments are also referred to as“soluble quenchers” or “soluble light emission modifiers”. In addition,without being bound by any particular theory, it is believed that alight emission modifier generally binds to nucleic acids in a lengthdependent manner. That is, light emission modifiers typically bind tolonger nucleic acids to a greater extent than to relatively shorternucleic acids. Accordingly, the extent to which a light emissionmodifier modifies the emission of light from a given labeled nucleicacid is typically proportional to the length of that nucleic acid. Forexample, if a labeled oligonucleotide is cleaved in a 5′-nucleasereaction, a particular light emission modifier will generally modify(e.g., quench, etc.) the emission of light from labeled fragments of theoligonucleotide to a lesser extent than from the intact oligonucleotide.Exemplary light emission modifiers include various diazine and thiazinesdyes, which are described further herein.

A “light-emitting labeling moiety” refers to a labeling moiety thatgenerates or is capable of generating detectable radiation or light.Certain light-emitting labeling moieties generate light, e.g., byfluorescence, chemiluminescence, bioluminescence, or the like.

A “mixture” refers to a combination of two or more different components.A “reaction mixture” refers a mixture that comprises molecules that canparticipate in and/or facilitate a given reaction. To illustrate, aamplification reaction mixture generally includes a solution containingreagents necessary to carry out an amplification reaction, and typicallycontains primers, a nucleic acid polymerase, dNTPs, and a divalent metalcation in a suitable buffer. A reaction mixture is referred to ascomplete if it contains all reagents necessary to carry out thereaction, and incomplete if it contains only a subset of the necessaryreagents. It will be understood by one of skill in the art that reactioncomponents are routinely stored as separate solutions, each containing asubset of the total components, for reasons of convenience, storagestability, or to allow for application-dependent adjustment of thecomponent concentrations, and that reaction components are combinedprior to the reaction to create a complete reaction mixture. Reactioncomponents may also be formulated in a dry form, e.g., tablets, and maythen be reconstituted prior to use. Furthermore, it will be understoodby one of skill in the art that reaction components are packagedseparately for commercialization and that useful commercial kits maycontain any subset of the reaction components which includes themodified primers of the invention.

A “moiety” or “group” refers to one of the portions into whichsomething, such as a molecule, is divided (e.g., a functional group,substituent group, or the like). For example, a probe may be consideredan oligonucleotide that optionally comprises a quencher moiety, alabeling moiety, or the like.

The term “nucleic acid” refers to a polymer of monomers that can becorresponded to a ribose nucleic acid (RNA) or deoxyribose nucleic acid(DNA) polymer, or analog thereof. This includes polymers of nucleotidessuch as RNA and DNA, as well as modified forms thereof, peptide nucleicacids (PNAs), locked nucleic acids (LNA™s), and the like. In certainapplications, the nucleic acid can be a polymer that includes multiplemonomer types, e.g., both RNA and DNA subunits. A nucleic acid can be orinclude, e.g., a chromosome or chromosomal segment, a vector (e.g., anexpression vector), an expression cassette, a naked DNA or RNA polymer,an amplicon, an oligonucleotide, a primer, a probe, etc. A nucleic acidcan be e.g., single-stranded or double-stranded. Unless otherwiseindicated, a particular nucleic acid sequence optionally comprises orencodes complementary sequences, in addition to any sequence explicitlyindicated.

A nucleic acid is typically single-stranded or double-stranded and willgenerally contain phosphodiester bonds, although in some cases, asoutlined herein, nucleic acid analogs are included that may havealternate backbones, including, for example and without limitation,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925 and thereferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al.(1988) J. Am. Chem. Soc. 110:4470; and Pauwels et al. (1986) ChemicaScripta 26:1419), phosphorothioate (Mag et al. (1991) Nucleic Acids Res.19:1437 and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.(1989) J. Am. Chem. Soc. 111:2321), O-methylphosphoroamidite linkages(Eckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press (1992)), and peptide nucleic acid backbones andlinkages (Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992)Chem. Int. Ed. Engl. 31:1008; Nielsen (1993) Nature 365:566; andCarlsson et al. (1996) Nature 380:207), which references are eachincorporated by reference. Other analog nucleic acids include those withpositively charged backbones (Denpcy et al. (1995) Proc. Natl. Acad.Sci. USA 92:6097); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghvi and P. Dan Cook; Mesmaeker et al. (1994)Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghvi and P. Dan Cook,which references are each incorporated by reference. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (Jenkins et al. (1995) Chem. Soc. Rev. pp169-176, which is incorporated by reference). Several nucleic acidanalogs are also described in, e.g., Rawls, C & E News Jun. 2, 1997 page35, which is incorporated by reference. These modifications of theribose-phosphate backbone may be done to facilitate the addition ofadditional moieties such as labeling moieties, or to alter the stabilityand half-life of such molecules in physiological environments.

In addition to naturally occurring heterocyclic bases that are typicallyfound in nucleic acids (e.g., adenine, guanine, thymine, cytosine, anduracil), nucleic acid analogs also include those having non-naturallyoccurring heterocyclic or other modified bases, many of which aredescribed, or otherwise referred to, herein. In particular, manynon-naturally occurring bases are described further in, e.g., Seela etal. (1991) Helv. Chim. Acta 74:1790, Grein et al. (1994) Bioorg. Med.Chem. Lett. 4:971-976, and Seela et al. (1999) Helv. Chim. Acta 82:1640,which are each incorporated by reference. To further illustrate, certainbases used in nucleotides that act as melting temperature (T_(m))modifiers are optionally included. For example, some of these include7-deazapurines (e.g., 7-deazaguanine, 7-deazaadenine, etc.),pyrazolo[3,4-d]pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl-dC,etc.), and the like. See, e.g., U.S. Pat. No. 5,990,303, entitled“SYNTHESIS OF 7-DEAZA-2′-DEOXYGUANOSINE NUCLEOTIDES,” which issued Nov.23, 1999 to Seela, which is incorporated by reference. Otherrepresentative heterocyclic bases include, e.g., hypoxanthine, inosine,xanthine; 8-aza derivatives of 2-aminopurine, 2,6-diaminopurine,2-amino-6-chloropurine, hypoxanthine, inosine and xanthine;7-deaza-8-aza derivatives of adenine, guanine, 2-aminopurine,2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine andxanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine;5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynylcytosine;5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil;5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil;5-ethynyluracil; 5-propynyluracil, and the like.

Additional examples of modified bases and nucleotides are also describedin, e.g., U.S. Pat. No. 5,484,908, entitled “OLIGONUCLEOTIDES CONTAINING5-PROPYNYL PYRIMIDINES,” issued Jan. 16, 1996 to Froehler et al., U.S.Pat. No. 5,645,985, entitled “ENHANCED TRIPLE-HELIX AND DOUBLE-HELIXFORMATION WITH OLIGOMERS CONTAINING MODIFIED PYRIMIDINES,” issued Jul.8, 1997 to Froehler et al., U.S. Pat. No. 5,830,653, entitled “METHODSOF USING OLIGOMERS CONTAINING MODIFIED PYRIMIDINES,” issued Nov. 3, 1998to Froehler et al., U.S. Pat. No. 6,639,059, entitled “SYNTHESIS OF[2.2.1]BICYCLO NUCLEOSIDES,” issued Oct. 28, 2003 to Kochkine et al.,U.S. Pat. No. 6,303,315, entitled “ONE STEP SAMPLE PREPARATION ANDDETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES,” issued Oct.16, 2001 to Skouv, and U.S. Pat. Application Pub. No. 2003/0092905,entitled “SYNTHESIS OF [2.2.1]BICYCLO NUCLEOSIDES,” by Kochkine et al.that published May 15, 2003, which are each incorporated by reference.

A “nucleotide” refers to an ester of a nucleoside, e.g., a phosphateester of a nucleoside. To illustrate, a nucleotide can include 1, 2, 3,or more phosphate groups covalently linked to a 5′ position of a sugarmoiety of the nucleoside.

A “nucleotide incorporating biocatalyst” refers to a catalyst thatcatalyzes the incorporation of nucleotides into a nucleic acid.Nucleotide incorporating biocatalysts are typically enzymes. An “enzyme”is a protein-based catalyst that acts to reduce the activation energy ofa chemical reaction involving other compounds or “substrates.” A“nucleotide incorporating enzyme” refers to an enzyme that catalyzes theincorporation of nucleotides into a nucleic acid. Exemplary nucleotideincorporating enzymes include, e.g., DNA polymerases, RNA polymerases,terminal transferases, reverse transcriptases, telomerases,polynucleotide phosphorylases, and the like. Other biocatalysts may beDNA-based (“DNAzymes”) or RNA-based (“ribozymes”). A “thermostableenzyme” refers to an enzyme that is stable to heat, is heat resistantand retains sufficient catalytic activity when subjected to elevatedtemperatures for selected periods of time. For example, a thermostablepolymerase retains sufficient activity to effect subsequent primerextension reactions when subjected to elevated temperatures for the timenecessary to effect denaturation of double-stranded nucleic acids.Heating conditions necessary for nucleic acid denaturation are wellknown in the art and are exemplified in U.S. Pat. No. 4,683,202,entitled “PROCESS FOR AMPLIFYING NUCLEIC ACID SEQUENCES,” issued Jul.28, 1987 to Mullis and U.S. Pat. No. 4,683,195, entitled “PROCESS FORAMPLIFYING, DETECTING, AND/OR-CLONING NUCLEIC ACID SEQUENCES,” issuedJul. 28, 1987 to Mullis et al., which are both incorporated byreference. As used herein, a thermostable polymerase is typicallysuitable for use in a temperature cycling reaction such as a PCR or a5′-nuclease reaction. For a thermostable polymerase, enzymatic activityrefers to the catalysis of the polymerization of the nucleotides in theproper manner to form primer extension products that are complementaryto a template nucleic acid.

An “oligonucleotide” or a “polynucleotide” refers to a nucleic acid thatincludes at least two nucleic acid monomer units (e.g., nucleotides),typically more than three monomer units, and more typically greater thanten monomer units. The exact size of an oligonucleotide generallydepends on various factors, including the ultimate function or use ofthe oligonucleotide. Oligonucleotides are optionally prepared by anysuitable method, including, but not limited to, isolation of an existingor natural sequence, DNA replication or amplification, reversetranscription, cloning and restriction digestion of appropriatesequences, or direct chemical synthesis by a method such as thephosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90-99;the phosphodiester method of Brown et al. (1979) Meth. Enzymol.68:109-151; the diethylphosphoramidite method of Beaucage et al. (1981)Tetrahedron Lett. 22:1859-1862; the triester method of Matteucci et al.(1981) J. Am. Chem. Soc. 103:3185-3191; automated synthesis methods; orthe solid support method of U.S. Pat. No. 4,458,066, entitled “PROCESSFOR PREPARING POLYNUCLEOTIDES,” issued Jul. 3, 1984 to Caruthers et al.,or other methods known in the art. All of these references areincorporated by reference.

The term “probe nucleic acid” or “probe” refers to a labeled orunlabeled oligonucleotide capable of selectively hybridizing to a targetor template nucleic acid under suitable conditions. Typically, a probeis sufficiently complementary to a specific target sequence contained ina nucleic acid sample to form a stable hybridization duplex with thetarget sequence under a selected hybridization condition, such as, butnot limited to, a stringent hybridization condition. A hybridizationassay carried out using the probe under sufficiently stringenthybridization conditions permits the selective detection of a specifictarget sequence. The term “hybridizing region” refers to that region ofa nucleic acid that is exactly or substantially complementary to, andtherefore hybridizes to, the target sequence. For use in a hybridizationassay for the discrimination of single nucleotide differences insequence, the hybridizing region is typically from about 8 to about 100nucleotides in length. Although the hybridizing region generally refersto the entire oligonucleotide, the probe may include additionalnucleotide sequences that function, for example, as linker binding sitesto provide a site for attaching the probe sequence to a solid support orthe like, as sites for hybridization of other oligonucleotides, asrestriction enzymes sites or binding sites for other nucleic acidbinding enzymes, etc. In certain embodiments, a probe of the inventionis included in a nucleic acid that comprises one or more labels (e.g., areporter dye, a quencher moiety, etc.), such as a 5′-nuclease probe, aFRET probe, a molecular beacon, or the like, which can also be utilizedto detect hybridization between the probe and target nucleic acids in asample. In some embodiments, the hybridizing region of the probe iscompletely complementary to the target sequence. However, in general,complete complementarity is not necessary (i.e., nucleic acids can bepartially complementary to one another); stable duplexes may containmismatched bases or unmatched bases. Modification of the stringentconditions may be necessary to permit a stable hybridization duplex withone or more base pair mismatches or unmatched bases. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001), which is incorporatedby reference, provides guidance for suitable modification. Stability ofthe target/probe duplex depends on a number of variables includinglength of the oligonucleotide, base composition and sequence of theoligonucleotide, temperature, and ionic conditions. One of skill in theart will recognize that, in general, the exact complement of a givenprobe is similarly useful as a probe. One of skill in the art will alsorecognize that, in certain embodiments, probe nucleic acids can also beused as primer nucleic acids. Exemplary probe nucleic acids include5′-nuclease probes, molecular beacons, among many others known topersons of skill in the art.

A “primer nucleic acid” or “primer” is a nucleic acid that can hybridizeto a target or template nucleic acid and permit chain extension orelongation using, e.g., a nucleotide incorporating biocatalyst, such asa polymerase under appropriate reaction conditions. A primer nucleicacid is typically a natural or synthetic oligonucleotide (e.g., asingle-stranded oligodeoxyribonucleotide, etc.). Although other primernucleic acid lengths are optionally utilized, they typically comprisehybridizing regions that range from about 8 to about 100 nucleotides inlength. Short primer nucleic acids generally utilize cooler temperaturesto form sufficiently stable hybrid complexes with template nucleicacids. A primer nucleic acid that is at least partially complementary toa subsequence of a template nucleic acid is typically sufficient tohybridize with the template for extension to occur. A primer nucleicacid can be labeled (e.g., a SCORPION® primer, etc.), if desired, byincorporating a label detectable by, e.g., spectroscopic, photochemical,biochemical, immunochemical, chemical, or other techniques. Toillustrate, useful labels include radioisotopes, fluorescent dyes,electron-dense reagents, enzymes (as commonly used in ELISAs), biotin,or haptens and proteins for which antisera or monoclonal antibodies areavailable. Many of these and other labels are described further hereinand/or otherwise known in the art. One of skill in the art willrecognize that, in certain embodiments, primer nucleic acids can also beused as probe nucleic acids.

A “quencher moiety” or “quencher” refers to a moiety that reduces and/oris capable of reducing the detectable emission of radiation, e.g.,fluorescent or luminescent radiation, from a source (“donor”) that wouldotherwise have emitted this radiation at a particular λ_(max). Aquencher typically reduces the detectable radiation emitted by thesource by at least 50%, typically by at least 80%, and more typically byat least 90%. Certain quenchers may re-emit the energy absorbed from,e.g., a fluorescent dye in a signal characteristic for that quencher andthus a quencher can also be a “label.” This phenomenon is generallyknown as fluorescent resonance energy transfer or FRET. Alternatively, aquencher may dissipate the energy absorbed from a fluorescent dye in aform other than light, e.g., as heat. Molecules commonly used in FRETinclude, for example, fluorescein, FAM, JOE, rhodamine, R6G, TAMRA, ROX,DABCYL, and EDANS. Whether a fluorescent dye is a donor or an acceptoris defined by its excitation and emission spectra, and the fluorescentdye with which it is paired. For example, FAM is most efficientlyexcited by light with a wavelength of 488 nm, and emits light with aspectrum of 500 to 650 nm, and an emission maximum of 525 nm. FAM is asuitable donor label for use with, e.g., TAMRA as a quencher, which hasat its excitation maximum 514 m-n. Exemplary non-fluorescent or darkquenchers that dissipate energy absorbed from a fluorescent dye includethe Black Hole Quenchers™ marketed by Biosearch Technologies, Inc.(Novato, Calif., USA), and the ECLIPSE® Dark Quenchers (EpochBiosciences, Bothell, Wash., USA). The Black Hole Quenchers™ (BHQ) arestructures comprising at least three radicals selected from substitutedor unsubstituted aryl or heteroaryl compounds, or combinations thereof,wherein at least two of the residues are linked via an exocyclic diazobond (see, e.g., International Publication No. WO 01/86001, entitled“DARK QUENCHERS FOR DONOR-ACCEPTOR ENERGY TRANSFER,” published Nov. 15,2001 by Cook et al., which is incorporated by reference). Exemplaryquenchers are also provided in, e.g., U.S. Pat. No. 6,465,175, entitled“OLIGONUCLEOTIDE PROBES BEARING QUENCHABLE FLUORESCENT LABELS, ANDMETHODS OF USE THEREOF,” which issued Oct. 15, 2002 to Horn et al.,which is incorporated by reference. Quenchers apply both to moleculesthat do not re-emit absorbed light as light of a longer wavelength(non-fluorescent) or by re-emitting light at a wavelength that isoutside the range that is detected (fluorescent).

In its broadest sense, a quencher refers to any molecule that is capableof reducing a light emission. It is noted that there are instances wherea quencher in not necessarily a FRET quencher. There is not arequirement that a quencher work by a strict “FRET” mechanism, andindeed, a quencher can function by any mechanism. There is norequirement for a spectral overlap between the fluorophore and thequencher. It is noted that quenching can include dynamic quenching(Forster, Dexter etc.), and static quenching (ground state complex).Quenching mechanisms can involve energy transfer, photoelectrontransfer, proton coupled electron transfer, dimer formation betweenclosely situated fluorophores, transient excited state interactions,collisional quenching, or formation of non-fluorescent ground statespecies. See, e.g., Principles of Fluorescence Spectroscopy, by JosephLakowicz; and Handbook of Fluorescent Probes by Richard Haugland.

A “sequence” of a nucleic acid refers to the order and identity ofnucleotides in the nucleic acid. A sequence is typically read in the 5′to 3′ direction.

A “single-labeled oligonucleotide” refers to an oligonucleotide thatincludes only one labeling moiety. In certain embodiments, the labelingmoiety is light emitting.

A substance is “soluble” when it is capable of being free in solution.For example, soluble light emission modifiers typically interactnon-covalently with nucleic acids when they are free in solution.

The terms “stringent” or “stringent conditions”, as used herein, denotehybridization conditions of low ionic strength and high temperature, asis well known in the art. See, e.g., Sambrook et al., Molecular Cloning:A Laboratory Manual, 3^(rd) Ed., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (2001); Current Protocols in Molecular Biology(Ausubel et al., ed., J. Wiley & Sons Inc., New York, 1997); Tijssen(1993), supra, each of which is incorporated by reference. Generally,stringent conditions are selected to be about 5-30° C. lower than thethermal melting point (T_(m)) for the specified sequence at a definedionic strength and pH. Alternatively, stringent conditions are selectedto be about 5-15° C. lower than the T_(m) for the specified sequence ata defined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength, pH and nucleic acid concentration) at which 50%of the probes complementary to the target hybridize to the targetsequence at equilibrium (as the target sequences are present in excess,at T_(m), 50% of the probes are hybridized at equilibrium).

A “subject” refers to an organism. Typically, the organism is amammalian organism, particularly a human organism. In certainembodiments, for example, a subject is a patient suspected of having agenetic disorder, disease state, or other condition.

A “subsequence,” “segment,” or “fragment” refers to any portion of anentire nucleic acid sequence.

A “thiazine dye” refers to any of a class of organic chemical compoundscontaining a tricyclic aromatic fused ring system, where two of thecarbons in the middle ring are replaced by a nitrogen atom and a sulfuratom. Exemplary thiazine dyes include methylene blue, methylene green,thionin, 1,9-dimethylmethylene blue, sym-dimethylthionin, toluidine blueO, new methylene blue, methylene violet bernthsen, azure A, azure B, andazure C.

The term “template nucleic acid” or “target nucleic acid” refers to anucleic acid that is to be amplified, detected, or otherwise analyzed.

Objects “thermally communicate” with one another when thermal energy istransferred or capable of being transferred between the objects. Incertain embodiments of the systems described herein, for example,thermal modulators thermally communicate with containers to modulatetemperature in the containers.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which onehalf of a population of double-stranded polynucleotides or nucleobaseoligomers (e.g., hybridization complexes), in homoduplexes orheteroduplexes, become dissociated into single strands. The predictionof a T_(m) of a duplex polynucleotide takes into account the basesequence as well as other factors including structural and sequencecharacteristics and nature of the oligomeric linkages. Methods forpredicting and experimentally determining T_(m) are known in the art.

For example, a T_(m) is traditionally determined by a melting curve,where a duplex nucleic acid molecule is heated in a controlledtemperature program, and the state of association/dissociation of thetwo single strands in the duplex is monitored and plotted until reachinga temperature where the two strands are completely dissociated. TheT_(m) is read from this melting curve. Alternatively, a T_(m) can bedetermined by an annealing curve, where a duplex nucleic acid moleculeis heated to a temperature where the two strands are completelydissociated. The temperature is then lowered in a controlled temperatureprogram, and the state of association/dissociation of the two singlestrands in the duplex is monitored and plotted until reaching atemperature where the two strands are completely annealed. The T_(m) isread from this annealing curve.

It is not intended that the invention be limited to any particularmethod for the determination of Tm. Methods for the experimentaldetermination of T_(m) are widely known in the art and are described ina variety of sources, e.g., Liew et al., “Genotyping ofSingle-Nucleotide Polymorphism by High-Resolution Melting of SmallAmplicons,” Clinical Chemistry 50(7):1156-1164 (2004); Reed and Wittwer“Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanningby High-Resolution Melting Analysis,” Clinical Chemistry50(10):1748-1754 (2004); Zhou et al., “Closed-Tube Genotyping withUnlabeled Oligonucleotide Probes and a Saturating DNA Dye,” ClinicalChemistry 50(8):1328-1335 (2004); and Zhou et al., “High-resolution DNAmelting curve analysis to establish HLA genotypic identity,” TissueAntigens 64:156-164 (2004). Melting/annealing curve analysisinstrumentation is commercially available from a variety ofmanufacturers.

As used herein, the term “sample” is used in its broadest sense, andrefers to any material subject to analysis. The term “sample” referstypically to any type of material of biological origin, for example, anytype of material obtained from animals or plants. A sample can be, forexample, any fluid or tissue such as blood or serum, and furthermore,can be human blood or human serum. A sample can be cultured cells ortissues, cultures of microorganisms (prokaryotic or eukaryotic), or anyfraction or products produced from or derived from biological materials(living or once living). Optionally, a sample can be purified, partiallypurified, unpurified, enriched or amplified. Where a sample is purifiedor enriched, the sample can comprise principally one component, e.g.,nucleic acid. More specifically, for example, a purified or amplifiedsample can comprise total cellular RNA, total cellular mRNA, cDNA, cRNA,or an amplified product derived there from.

The sample used in the methods of the invention can be from any source,and is not limited. Such sample can be an amount of tissue or fluidisolated from an individual or individuals, including, but not limitedto, for example, skin, plasma, serum, whole blood, blood products,spinal fluid, saliva, peritoneal fluid, lymphatic fluid, aqueous orvitreous humor, synovial fluid, urine, tears, blood cells, bloodproducts, semen, seminal fluid, vaginal fluids, pulmonary effusion,serosal fluid, organs, bronchio-alveolar lavage, tumors, paraffinembedded tissues, etc. Samples also can include constituents andcomponents of in vitro cell cultures, including, but not limited to,conditioned medium resulting from the growth of cells in the cellculture medium, recombinant cells, cell components, etc.

As used herein, the expression “hepatitis C virus type” refers to thecategorization of a hepatitis C virus (HCV) based on its genomicorganization. The categorization of an HCV isolate into a particulartype category reflects its genomic relatedness to other HCV isolates andits relatively lesser relatedness to other HCV isolates. As used herein,HCV typing nomenclature is consistent with the widely adoptednomenclature proposed by Simmonds et al (1994) Letter, Hepatology19:1321-1324. See, also, Zein (2000) “Clinical Significance of HepatitisC Virus Genotypes,” Clinical Microbiol. Reviews 13(2):223-235; Maertensand Stuyver (1997) “Genotypes and Genetic Variation of Hepatitis CVirus,” p. 182-233, In Harrison, and Zuckerman (eds.), The MolecularMedicine of Viral Hepatitis, John Wiley & Sons, Ltd., Chichester,England.). The system of Simmonds et al (1994) places the known HCVisolates into one of eleven (11) HCV genotypes, namely genotypes 1through 11. Each genotype is further subdivided into groupings termedsubtypes that reflect relatedness among strains of the same genotype. AnHCV subtype is written by a lowercase roman letter following thegenotype, e.g., subtype 1a, subtype 1c, subtype 6a, etc. Geneticvariants found within an individual isolate are termed quasispecies.Approximately 78 HCV subtypes encompassing all 11 genotypes are knownworldwide; the number of subtypes is not static; as more HCV isolatesare studied and sequenced, it is likely that additional subtypes (andpossibly genotypes) may be recognized. As used herein, the term “virustypes” can refer to either genotypes or subtypes.

Some reports (see, e.g., Robertson et al., (1998) Arch. Virol.,143(12):2493-2503) suggest that viral genomic organization is bestrepresented by the creation of viral clades, reflecting the observationthat some HCV genotypes are more closely related to each other than toother HCV genotypes. In this system, clades 1, 2, 4 and 5 correspond togenotypes 1, 2, 4 and 5, while lade 3 comprises genotypes 3 and 10, andclade 6 comprises genotypes 6, 7, 8, 9 and 11. The description of thepresent invention does not use the lade nomenclature.

Additional detailed description of HCV genotypes and genotypingmethodologies is found in cofiled U.S. patent application Ser. No.11/474,125, filed on Jun. 23, 2006, entitled “PROBES AND METHODS FORHEPATITIS C VIRUS TYPING USING SINGLE PROBE ANALYSIS,” by Gupta andWill; and also in cofiled U.S. patent application Ser. No. 11/474,092,filed on Jun. 23, 2006, entitled “PROBES AND METHODS FOR HEPATITIS CVIRUS TYPING USING MULTIDIMENSIONAL PROBE ANALYSIS,” by Gupta and Will.The entire content of these two cofiled applications are herebyincorporated by reference in their entirety for all purposes.

As used herein, the expression “derived from” refers to a component thatis isolated from or made using a specified sample, molecule, organism orinformation from the specified molecule or organism. For example, anucleic acid molecule that is derived from a hepatitis C virus can be,for example, a molecule of the HCV genome, or alternatively, atranscript from the HCV genome, or alternatively, a synthetic nucleicacid comprising a polynucleotide sequence that corresponds to an HCVpolynucleotide sequence.

As used herein, the term “monitor” refers to periodic or continuoussurveillance, testing, data collecting and/or quantitation. Monitoringcan be automated, and the information (e.g., a dataset) gathered duringthe monitoring can be printed or can be compiled as a computer readableand/or computer storable format.

As used herein, the term “correlate” refers to making a relationshipbetween two or more variables, values or entities. If two variablescorrelate, the identification of one of those variables can be used todetermine the value of the remaining variable.

As used herein, the term “kit” is used in reference to a combination ofarticles that facilitate a process, method, assay, analysis ormanipulation of a sample. Kits can contain written instructionsdescribing how to use the kit (e.g., instructions describing the methodsof the present invention), chemical reagents or enzymes required for themethod, primers and probes, as well as any other components.

As used herein, the expression “asymmetric PCR” refers to thepreferential PCR amplification of one strand of a DNA target byadjusting the molar concentration of the primers in a primer pair sothat they are unequal. An asymmetric PCR reaction produces apredominantly single-stranded product and a smaller quantity of adouble-stranded product as a result of the unequal primerconcentrations. As asymmetric PCR proceeds, the lower concentrationprimer is quantitatively incorporated into a double-stranded DNAamplicon, but the higher concentration primer continues to prime DNAsynthesis, resulting in continued accumulation of a single strandedproduct.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B schematically illustrate an assay that includes lightemission modifiers according to one embodiment of the invention.

FIGS. 2 A and B schematically illustrate another assay that includeslight emission modifiers according to one embodiment of the invention.

FIG. 3 is a block diagram showing a representative system according toone embodiment of the invention.

FIG. 4 is a graph (ordinate represents percent fluorescence, abscissarepresents the new methylene blue concentration (μg/mL)) that showsfluorescence quenching of single-stranded (ss) and double-stranded (ds)DNA and thymidine dimer with increasing amounts of new methylene blue inseparate reverse transcription-polymerase chain (RT-PCR) mixtures.

FIG. 5 is a graph (ordinate represents percent fluorescence, abscissarepresents light emission modifier concentration (μg/mL)) that showsfluorescence quenching of ss DNA with increasing amounts of sixdifferent thiazine dyes in a cocktail containing all the components ofan RT-PCR mixture.

FIG. 6 is a graph (ordinate represents percent fluorescence, abscissarepresents light emission modifier concentration (μg/mL)) that showsfluorescence quenching of ds DNA with increasing amounts of sixdifferent thiazine dyes in a cocktail containing all the components ofan RT-PCR mixture.

FIG. 7 is a graph (ordinate represents percent fluorescence, abscissarepresents light emission modifier concentration (μg/mL)) that showsfluorescence quenching of dinucleotide DNA with increasing amounts ofsix different thiazine dyes in a cocktail containing all the componentsof an RT-PCR mixture.

FIG. 8 is a graph (ordinate represents percent fluorescence, abscissarepresents light emission modifier concentration (μg/mL)) that showsfluorescence quenching of ss DNA with increasing amounts of methyleneblue in a cocktail containing all the components of an RT-PCR mixture,with or without poly rA.

FIG. 9 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that showspolymerase chain reaction (PCR) detection of hepatitis C virus (HCV) DNAwith a single-labeled HCV probe in the presence of variousconcentrations of azure B.

FIG. 10 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that showsfluorescence as a function of cycle number using a single-labeled HCVprobe in human immunodeficiency virus (HIV) kinetic PCR reactions in thepresence of various concentrations of azure B.

FIG. 11 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of azure B.

FIG. 12 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of new methylene blue.

FIG. 13 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of new methylene blue.

FIG. 14 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of 1,9 dimethyl methylene blue.

FIG. 15 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of 1,9 dimethyl methylene blue.

FIG. 16 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of azure A.

FIG. 17 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of azure C.

FIG. 18 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of thionin.

FIG. 19 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of methylene green.

FIG. 20 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows acomparison of azure A, azure B, and azure C in the PCR detection of HCVDNA with a single-labeled probe in the presence of 40 μg/mLconcentrations of the azure dye.

FIG. 21 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows acomparison of azure A, azure B, azure C, methylene blue, toluidine blue,thionin, and methylene green in the PCR detection of HCV DNA with asingle-labeled probe in the presence of 40 μg/mL concentrations of thethiazine dye.

FIG. 22 is a photograph of a polyacrylamide gel that shows an analysisof HCV PCR reactions with 20,000 copies of HCV DNA, various probes, andvarious amounts of methylene blue. Panels A and B represent duplicatereactions.

FIG. 23 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows PCRdetection of IQS (internal quantitation standard) DNA with a HEX labeledsingle-labeled probe in the presence of various concentrations ofmethylene blue.

FIG. 24 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows thesimultaneous PCR detection of HCV and IQS DNA with a combination of FAM-and HEX-labeled single-labeled probes in the presence of variousconcentrations of methylene blue.

FIG. 25 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows acomparison of signals obtained from a single-labeled probe and adual-labeled probe in the absence of a light emission modifier. Theassays were performed either in the presence or in the absence ofcarrier nucleic acid, poly rA.

FIG. 26 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows acomparison of signals obtained from a single-labeled probe and adual-labeled probe in the presence of 30 μg/mL of a light emissionmodifier, azure B. The assays were performed either in the presence orin the absence of carrier nucleic acid, poly rA.

FIG. 27 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows acomparison of signals obtained from a single-labeled probe and adual-labeled probe in the presence of 30 μg/mL of a light emissionmodifier, azure B. The assays were performed either in the presence orin the absence of carrier nucleic acid, poly rA.

FIG. 28 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from DNA template titrations that included a single-labeledprobe and methylene blue.

FIG. 29 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from RNA template titrations that included a single-labeledprobe and methylene blue.

FIG. 30 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows acomparison of signals obtained from a single-labeled probe and adual-labeled probe in the presence of 40 μg/mL of a light emissionmodifier, methylene blue in the detection of 2-200,000 input copies ofHIV DNA.

FIG. 31 is a photograph of a polyacrylamide gel that shows an analysisof HCV and HIV PCR reactions with 2-200,000 input copies of DNA, in thepresence of 40 μg/mL methylene blue. Panels A and B represent HCV andHIV reactions, respectively.

FIG. 32 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of new methylene blue when fluorescence ismeasured at 40° C.

FIG. 33 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows PCRdetection of HCV DNA with a single-labeled probe in the presence ofvarious concentrations of new methylene blue, when fluorescence ismeasured at 40° C.

FIG. 34 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows acomparison of PCR detection of HCV DNA with a single-labeled probe inthe presence of 40 μg/mL new methylene blue, when fluorescence ismeasured at two different temperatures, 58° C., or 40° C.

FIG. 35 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-BHQ dual-labeled probe and various amounts of methylene blue.

FIG. 36 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV withtwice the amount of a FAM-BHQ dual-labeled probe than was used in theassays represented in FIG. 35 and various amounts of methylene blue.

FIG. 37 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows acomparison of the baseline fluorescence levels obtained in 5′-nucleaseassays detecting HCV with two different levels of a FAM-BHQ dual-labeledprobe and various amounts of methylene blue.

FIG. 38 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows relativefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-BHQ dual-labeled probe and various amounts of methylene blue.

FIG. 39 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-BHQ dual-labeled probe and various amounts of dimethyl methyleneblue.

FIG. 40 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows relativefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-BHQ dual-labeled probe and various amounts of dimethyl methyleneblue.

FIG. 41 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-BHQ dual-labeled probe and various amounts of new methylene blue.

FIG. 42 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows relativefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-BHQ dual-labeled probe and various amounts of new methylene blue.

FIG. 43 is an amplification plot (ordinate represents raw fluorescence,abscissa represents the cycle number) that shows baseline fluorescencelevels obtained in 5′-nuclease assays detecting IQS DNA with a HEX-CY5dual-labeled probe and various amounts of methylene blue.

FIG. 44 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows relativefluorescence levels obtained in 5′-nuclease assays detecting IQS DNAwith a HEX-CY5 dual-labeled probe and various amounts of methylene blue.

FIG. 45 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-CY5 dual-labeled probe and various amounts of Janus Green B.

FIG. 46 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-CY5 dual-labeled probe and various amounts of toluidine blue.

FIG. 47 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-CY5 dual-labeled probe and various amounts of Victoria Pure Blue BO.

FIG. 48 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-CY5 dual-labeled probe and various amounts of azure A.

FIG. 49 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-CY5 dual-labeled probe and various amounts of methylene green.

FIG. 50 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-CY5 dual-labeled probe and various amounts of thionin.

FIG. 51 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows baselinefluorescence levels obtained in 5′-nuclease assays detecting HCV with aFAM-CY5 dual-labeled probe and various amounts of azure B.

FIG. 52A provides the nucleotide sequence of a FAM-labeled HCV-specificprobe. FIG. 52B provides a melting curve (Tm) analysis showing rawfluorescence plotted as a function of temperature using thesingle-labeled HCV genotyping probe shown in FIG. 52A and syntheticnucleic acid targets. The melting reaction does not include a solublequencher. Fluorescence was measured using an excitation filter at 485 nmwith a 20 nm bandwidth, and an emission filter at 520 nm with a 10 nmbandwidth. The results of the seven separate experiments are overlaid onthe same graph. A representative set of data is shown.

FIG. 53 provides a melting curve analysis showing raw fluorescenceplotted as a function of temperature using a single-labeled HCVgenotyping probe and synthetic nucleic acid targets. The experimentalconditions were identical to those used in FIG. 52B, except that 10μg/mL of soluble light emission modifier (i.e., soluble quencher)methylene blue were included in the reactions.

FIG. 54 provides a melting curve analysis using the same conditions usedin FIG. 53, except that 20 μg/mL of soluble quencher methylene blue wereincluded in the reactions.

FIG. 55 provides the first derivative plot of the melting curve analysisshown in FIG. 54. The HCV genotypes and the experimentally observed Tmvalues are indicated.

FIG. 56 provides first derivative plot of a melting curve (Tm) analysisusing a FAM single-labeled HCV genotyping probe, a synthetic nucleicacid target corresponding to HCV genotype 1a/b and four increasingconcentrations of methylene blue. The sequences of the probe andsynthetic template are shown, which form a perfect match duplex. Theresults of the four separate experiments are overlaid on the same graph.A representative set of data is shown.

FIG. 57 provides a first derivative plot of a melting curve (Tm)analysis using a FAM single-labeled HCV genotyping probe, a syntheticnucleic acid target corresponding to HCV genotype 6 and four increasingconcentrations of methylene blue. The sequences of the probe andsynthetic template are shown, which form a duplex with one mismatch. Theresults of the four separate experiments are overlaid on the same graph.A representative set of data is shown.

FIG. 58 provides first derivative plot of a melting curve (Tm) analysisusing a FAM single-labeled HCV genotyping probe, a synthetic nucleicacid target corresponding to HCV genotype 5 and four increasingconcentrations of methylene blue. The sequences of the probe andsynthetic template are shown, which form a duplex with two mismatchpositions. The results of the four separate experiments are overlaid onthe same graph. A representative set of data is shown.

FIG. 59 provides first derivative plot of a melting curve (Tm) analysisusing a FAM single-labeled HCV genotyping probe, a synthetic nucleicacid target corresponding to HCV genotype 2a/c and four increasingconcentrations of methylene blue. The sequences of the probe andsynthetic template are shown, which form a duplex with three mismatchpositions. The results of the four separate experiments are overlaid onthe same graph. A representative set of data is shown.

FIG. 60 provides a bar graph summary of Tm determinations using the HCVprobes indicated with the various HCV synthetic templates shown. The Tmdeterminations were made using various concentrations of new methyleneblue soluble quencher, as indicated. In one set of Tm determinations, anon-labeled probe was used in conjunction with SYBR® Green indicator. Arepresentative set of data is shown.

FIG. 61 provides a bar graph summary of Tm determinations using the HCVprobe provided in FIG. 52A and engineered synthetic templates thatcontain single base mismatches in various mismatch combinations. The Tmdeterminations were made using two different concentrations of methyleneblue soluble quencher, as indicated. Also shown in the bar graph are thepredicted Tm values of the various duplexes (in the absence of methyleneblue) generated by Visual OMP software (DNA Software, Inc., Ann Arbor,Mich.).

FIG. 62 provides nucleotide sequences corresponding to or derived fromthe HIV genome that find use with the invention. The sequences includethe SK145 forward HIV amplification primer region, the reversecomplement of the GAG152 reverse amplification primer region, and thereverse complement of the HIV GAG108FBHQ29I 5′-nuclease quantitationprobe region. Beneath these sequences are the corresponding homologousdomains from known HIV subtype isolates. Variable positions areindicated.

FIG. 63 provides a graph with the results of HIV RNA amplification(RT-PCR) quantitation using the SK145BU and GAG152BU amplificationprimers and the GAG108FBHQ29I 5′-nuclease quantitation probe. VariousHIV RNA templates (106 copies each) are used in the amplificationreactions, as shown. No thiazine dye is present in the reactions. Theresults of each genotype analysis are overlaid on the same graph. Arepresentative set of data is shown. The C_(T) number for the variousHIV genotypes tested is provided.

FIG. 64 provides a graph with the results of an HIV RNA amplificationquantitation analysis identical to that described in FIG. 63, exceptthat each of the reactions also contains 50 μg/mL of new methylene blue.

FIG. 65 provides a graph with the results of HIV RNA amplification(RT-PCR) quantitation using the SK145BU and GAG152BU amplificationprimers and the GAG108FBHQ29I 5′-nuclease quantitation probe. An RNAtemplate corresponding to HIV genotype 110-5 is used in theamplification reaction (10⁶ copies). This genotype results in a total ofsix mismatches under the forward primer and one mismatch under the5′-nuclease quantitation probe. Increasing concentrations of newmethylene blue are used in the reactions. The results of each analysisare overlaid on the same graph. A representative set of data is shown.The C_(T) number for each of the various amplification reactions isprovided.

FIG. 66 provides a graph with the results of HIV RNA amplification(RT-PCR) quantitation using the SK145BU and GAG152BU amplificationprimers. An RNA template corresponding to HIV genotype 110-5 is used inthe amplification reaction (10⁶ copies). Amplicon detection andquantitation is made by the addition of SYBR® Green to the reaction.Different concentrations of Molecular Probes SYBR® Green (1× which is a1:10,000 dilution of the stock SYBR® dye solution, and 4× which is a1:2500 dilution of the stock dye solution) and new methylene blue (0-50μg/mL) are used in the reactions. The results of each analysis areoverlaid on the same graph. A representative set of data is shown. TheC_(T) number observed for each reaction is indicated.

DETAILED DESCRIPTION Introduction

The present invention provides simple and robust methods and otheraspects related to the modulation of light emissions from labelednucleic acids. To illustrate, labeled nucleic acids, such as 5′-nucleaseprobes, molecular beacons, SCORPIONS primers, fluorescence resonanceenergy transfer (FRET) probes, etc. are commonly used to detect nucleicacids in various applications, including genotyping, diagnostics,forensics, among many others well known to those of skill in the art.Many of these probes include light-emitting labeling moieties, such asfluorescent reporter dyes, and quencher moieties that reduce thedetectable emission of light from the light-emitting moieties when thetwo moieties are in suitable proximity to one another. Although thesequencher moieties reduce detectable light emissions, this reduction isoften incomplete. That is, these multiply labeled probes frequently havean associated residual or baseline emission of light. As the amount ofprobe is increased in a reaction mixture, whether due to the use ofmultiple sets of different probes in multiplexing applications, orhigher amounts of a given probe in essentially any application, thisbaseline light emission also tends to increase. Baseline light emissionssuch as these typically negatively impact the performance of assaysinvolving these probes by, e.g., limiting the sensitivity (i.e., theability of the assay to discriminate between small differences inanalyte concentration) and dynamic range (i.e., the useful range of theassay which extends from the lowest concentration at which quantitativemeasurements can be made (limit of quantitation, or LOQ) to theconcentration at which the calibration curve departs from linearity(limit of linearity, LOL) of detection). Thus, certain light emissionmodifiers described herein are used to further reduce, if not eliminate,these baseline light emissions in some embodiments, to improve theperformance of assays involving these types of labeled probes.

In addition to providing approaches to modulating light emissions fromprobes that comprise multiple labels, such as those having pairs ofreporter and quencher moieties, the invention also provides for themodulation of light emissions from probes that each include only asingle light-emitting moiety. These approaches can also be used toeffect the real-time detection of target nucleic acids, includingreal-time reverse transcription-polymerase chain reaction-based (kineticRT-PCR) assays with signal dynamic ranges that are suitable for highlysensitive detection. Similar to other multiplexing approaches describedherein, in certain embodiments a single type of light emission modifiercan be used to quench multiple single-labeled probes that have differentlight-emitting moieties in the same reaction mixture to effect thesimultaneous detection of multiple target nucleic acids. Moreover,single-labeled probes are typically easier to synthesize and less costlyto produce than multi-labeled probes.

In overview, the invention provides reaction mixtures that includelight-emitting labeled oligonucleotides (e.g., 5′-nuclease probes, etc.)and light emission modifiers (e.g., soluble light emission modifiers)that modify the emission of light from the oligonucleotides. Exemplarylight emission modifiers include a variety of diazine and thiazine dyes.In certain embodiments, these reaction mixtures, or components thereof,are included in kits. Methods of modifying the emission of light fromlabeled oligonucleotides, e.g., as part of nucleic acid amplificationassays in which target nucleic acids are detected in real-time are alsoprovided. In addition, systems for detecting light emitted from thelabeled oligonucleotides in the reaction mixtures described herein arealso provided. These and a variety of other aspects and features of thepresent invention will be apparent upon a complete review of thisdisclosure.

To illustrate, FIGS. 1A and B schematically show an assay in which lightemission modifiers are used to substantially quench light emissions froma 5′-nuclease probe that is labeled with a single light-emitting moiety(e.g., a fluorescent dye, etc.). As shown in FIG. 1A, a reaction mixtureincludes target nucleic acid 100, primer 102, probe 104, and polymerase110 (having a 5′ to 3′ nuclease activity). Fluorophore 106 is covalentlyattached at or near a 5′ terminus of probe 104. As further shown, thereaction mixture also includes light emission modifier 108, whichnon-covalently associates with target nucleic acid 100 and primer 102.Light emission modifier 108 also non-covalently associates with probe104 to substantially quench fluorescence emitted from probe 104. Asshown in FIG. 1B, as the assay proceeds, polymerase 110 cleavesfragments from probe 104, which is bound to target nucleic acid 100. Inthis process, a fragment that comprises fluorophore 106 is released fromthe remaining portion of probe 104. As a consequence, a detectableincrease in fluorescence results, since the fluorescence emitted byfluorophore 106 from the fragment is less quenched than from probe 104prior to cleavage. That is, the light emission modifiers describedherein typically quench or reduce light emissions from labeled nucleicacids in a length dependent manner.

To further illustrate, FIGS. 2 A and B schematically depict an assay inwhich light emission modifiers are used to reduce baseline lightemissions from a dual labeled 5′-nuclease probe. As shown in FIG. 2A, areaction mixture includes target nucleic acid 200, primer 202, probe204, and polymerase 212 (having a 5′ to 3′ nuclease activity).Fluorophore 208 is covalently attached at a 5′ terminus of probe 204 andquencher 206 is covalently attached at a 3′ terminus of probe 204.Although not shown here, fluorophore 208 or quencher 206 may optionallybe attached to internal residues of probe 204. As further shown, thereaction mixture also includes light emission modifier 210, whichnon-covalently associates with target nucleic acid 200 and primer 202.Light emission modifier 210 also non-covalently associates with probe204 to reduce baseline fluorescence emitted from probe 204. As shown inFIG. 2B, as the assay proceeds, polymerase 212 cleaves fragments fromprobe 204, which is bound to target nucleic acid 200. Similar to theprocess described above with respect to FIGS. 1A and B, a fragment thatcomprises fluorophore 208 is released and a detectable increase influorescence results.

Reaction Mixtures

The reaction mixtures of the invention can be used in a wide variety ofapplications where it is desirable to modify the emission of light fromlabeled nucleic acids. In some embodiments, for example, the reactionmixtures described herein are utilized in performing homogeneousamplification/detection assays (e.g., real-time PCR, etc.), particularlyin multiplex formats in which multiple labeled probes are pooledtogether. Certain of the light emission modifiers described hereinreduce the baseline emission of light from labeled probes under thevaried temperature and other reaction conditions typically used in thesetypes of assays unlike many previously known compounds. In addition tolight emission modifiers and labeled oligonucleotides, other reagentsthat are optionally included in the reaction mixtures of the inventionare described in greater detail below.

Light Emission Modifiers

The light emission modifiers used in the reaction mixtures and otheraspects of the invention typically include a variety of properties thatmake them well suited to modulate or modify emissions of light fromlabeled probes in various types of nucleic acid amplification reactionsand assays. To illustrate, these light emission modifiers typically bindto both single-stranded nucleic acids (e.g., single-stranded probes) andto double-stranded nucleic acids (e.g., single-stranded probeshybridized to target nucleic acids). Further, without being bound by anyparticular theory, it is believed that the light emission modifiersdescribed herein generally bind to nucleic acids and modify lightemission from labels associated with the nucleic acids in a lengthdependent manner. That is, the extent that a light emission modifiermodifies the emission of light from a given labeled oligonucleotide istypically proportional to the length of that oligonucleotide. Forexample, a particular light emission modifier will generally modify theemission of light from labeled fragments of the oligonucleotide to alesser extent than from the intact or full-length oligonucleotide, e.g.,prior to cleavage in a 5′-nuclease reaction. A given light emissionmodifier is also typically able to effectively modify the emission oflight from a variety of different light-emitting moieties. In otherwords, the modifications (e.g., quenching) effected by these lightemission modifiers are generally spectral overlap independent oruniversal and without being bound to any particular theory of operation,likely occur by way of ground state complex formation. This is animportant property, for example, in multiplexing assays in whichmultiple probes labeled with different fluorophores or other labelingmoieties are commonly utilized.

To further illustrate, the light emission modifiers of the inventiongenerally remain bound to, and modify light emissions from, e.g.,full-length probes at temperatures commonly used in, e.g., kinetic PCRmonitoring (e.g., annealing temperatures of between about 35° C. toabout 60° C., extension temperatures of between about 65° C. to about80° C., anneal-extend step temperatures of between about 35° C. to about80° C. for two-step PCRs, etc.). Suitable PCR reaction conditions arealso described below and in, e.g., Gelfand et al. (Eds.), PCRApplications: Protocols for Functional Genomics, Elsevier Science &Technology Books (1999), which is incorporated by reference. Moreover,the light emission modifiers of the invention have the ability to bindto, and modify light emissions from, full-length probes in reactionmixtures that include various other PCR components, such as buffers,salts, metal ions, primers, dNTPs, ddNTPs or other terminatornucleotides, glycerol, DMSO, poly rA, and the like. The light emissionmodifiers described herein also generally do not appreciably interferewith any of the steps used in PCR (e.g., annealing, extension,denaturing). In RT-PCR applications, the light emission modifiersdescribed herein also typically do not significantly inhibit reversetranscription steps. An additional advantage of these light emissionmodifiers is that they continue to modify the emission of light fromfull-length probes even in the presence of large amounts of accumulatingamplicons with little partitioning to these PCR products. To furtherexemplify, the light emission modifiers described herein also do nothave sufficient, if any, intrinsic fluorescence in certain regions ofvisible spectrum that might otherwise interfere with or bias assaydetection. Many of these attributes are also illustrated in the examplesprovided below or otherwise referred to herein.

Many different light emission modifiers are suitable for use in thereaction mixtures and other aspects of the invention. Typically, lightemission modifiers are soluble nucleic acid binding compounds that arecapable of modifying the emission of light from labeledoligonucleotides, such as 5′-nuclease probes, molecular beacons, or thelike, at reaction temperatures commonly used in performing real-time PCRreaction steps, such as at annealing temperatures of at least about 40°C., etc. In some embodiments, for example, the light emission modifiersof the invention include various diazine and thiazine dyes. Exemplarydiazine dyes that can be used as light emission modifiers include, e.g.,azocarmine dyes (e.g., azocarmine A, azocarmine B (C₂₈H₁₇N₃O₉S₃Na₂),azocarmine G (C₂₈H₁₈N₃O₆S₂Na), etc.), phenazine dyes,diethylsafraninazodimethylaniline chloride (i.e., Janus Green B orDiazine Green 5 (C₃₀H₃₁N₆Cl)), and the like. The chemical structures ofsome of these diazine dyes are presented in Table I.

TABLE I AZOCARMINE G

CELESTINE BLUE JANUS GREEN B

To further illustrate, exemplary thiazine dyes that can be used as lightemission modifiers include, e.g., methylene blue (C₁₆H₁₈ClN₃S),methylene green (C₁₆H₁₇ClN₄O₂S), thionin (C₁₂H₁₀ClN₃S),sym-dimethylthionin, toluidine blue O (C₁₅H₁₆N₃SCl), new methylene blue(C₁₈H₂₂ClN₃S), methylene violet bernthsen, azure A (C₁₄H₁₄ClN₃S), azureB (C₁₅H₁₆ClN₃S), azure C(C₁₃H₁₂ClN₃S), and the like. The chemicalstructures of some of these thiazine dyes are presented in Table II.

TABLE II THIONIN

AZURE C

AZURE A

AZURE B

SYM-DIMETHYLTHIONIN

METHYLENE VIOLET BERNTHSEN

METHYLENE BLUE

1,9-DIMETHYLMETHYLENE BLUE

NEW METHYLENE BLUE

TOLUIDINE BLUE O

METHYLENE GREEN

The amount of the particular light emission modifier included in a givenreaction mixture typically depends on the extent of modification sought.Typically, the extent of light emission modification is proportional tothe amount of light emission modifier present in a reaction mixture.Although other quantities are optionally utilized, light emissionmodifiers are typically present at between about 5 μg/mL of the reactionmixture and about 100 μg/mL of the reaction mixture, more typically atbetween about 10 μg/mL of the reaction mixture and about 75 μg/mL of thereaction mixture, and still more typically at between about 15 μg/mL ofthe reaction mixture and about 50 μg/mL of the reaction mixture (e.g.,about 20 μg/mL, about 30 μg/mL, about 40 μg/mL, etc.). In someembodiments, reaction mixtures include light emission modifierconcentrations that are in excess of amplicon concentrations. Theeffects of various light emission modifier concentrations in reactionmixtures are further illustrated in the examples provided below. In someembodiments, more than one light emission modifier can be used in thesame reaction mixture. In these embodiments, the different lightemission modifiers are optionally present at the same or at differentconcentrations in the particular reaction mixture. As one example, areaction mixture may include 20 μg of new methylene blue per mL of thereaction mixture and 30 μg of methylene blue per mL of the reactionmixture. Light emission modifiers are readily available from variouscommercial suppliers including, e.g., Sigma-Aldrich Corp. (St. Louis,Mo., USA).

Labeled Oligonucleotides

The reaction mixtures of the invention include labeled oligonucleotidesin addition to light emission modifiers. Various approaches can beutilized by one of skill in the art to design oligonucleotides for useas probes (e.g., 5′-nuclease probes, molecular beacons, FRET probes,etc.) and/or primers. To illustrate, the DNAstar software packageavailable from DNASTAR, Inc. (Madison, Wis., U.S.A.) can be used forsequence alignments. For example, target nucleic acid sequences andnon-target nucleic acid sequences can be uploaded into DNAstar EditSeqprogram as individual files, e.g., as part of a process to identifyregions in these sequences that have low sequence similarity. To furtherillustrate, pairs of sequence files can be opened in the DNAstarMegAlign sequence alignment program and the Clustal W method ofalignment can be applied. The parameters used for Clustal W alignmentsare optionally the default settings in the software. MegAlign typicallydoes not provide a summary of the percent identity between twosequences. This is generally calculated manually. From the alignments,regions having, e.g., less than a selected percent identity with oneanother are typically identified and oligonucleotide sequences in theseregions can be selected. Many other sequence alignment algorithms andsoftware packages are also optionally utilized. Sequence alignmentalgorithms are also described in, e.g., Notredame et al. (2000)“T-coffee: a novel method for fast and accurate multiple sequencealignment,” J. Mol. Biol. 302:205-217, Edgar (2004) “MUSCLE: a multiplesequence alignment method with reduced time and space complexity,” BMCBioinformatics 5:113, Mount, Bioinformatics: Sequence and GenomeAnalysis, Cold Spring Harbor Laboratory Press (2001), and Durbin et al.,Biological Sequence Analysis: Probabilistic Models of Proteins andNucleic Acids, Cambridge University Press (1998), which are eachincorporated by reference.

To further illustrate, optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman(1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm ofNeedleman & Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson & Lipman (1988) Proc. Nat'l. Acad. Sci. USA85:2444, which are each incorporated by reference, and by computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (Madison, Wis., U.S.A.)), or by even by visual inspection.

Another example algorithm that is suitable for determining percentsequence identity is the BLAST algorithm, which is described in, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410, which is incorporatedby reference. Software for performing versions of BLAST analyses ispublicly available through the National Center for BiotechnologyInformation on the world wide web at ncbi.nlm.nih.gov/ as of Jun. 30,2005.

An additional example of a useful sequence alignment algorithm isPILEUP. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle (1987) J. Mol. Evol. 35:351-360, which isincorporated by reference.

Oligonucleotide probes and primers are optionally prepared usingessentially any technique known in the art. In certain embodiments, forexample, the oligonucleotide probes and primers are synthesizedchemically using essentially any nucleic acid synthesis method,including, e.g., the solid phase phosphoramidite method described byBeaucage and Caruthers (1981) Tetrahedron Letts. 22(20):1859-1862, whichis incorporated by reference. To further illustrate, oligonucleotidescan also be synthesized using a triester method (see, e.g., Capaldi etal. (2000) “Highly efficient solid phase synthesis of oligonucleotideanalogs containing phosphorodithioate linkages” Nucleic Acids Res.28(9):e40 and Eldrup et al. (1994) “Preparation ofoligodeoxyribonucleoside phosphorodithioates by a triester method”Nucleic Acids Res. 22(10):1797-1804, which are both incorporated byreference). Other synthesis techniques known in the art can also beutilized, including, e.g., using an automated synthesizer, as describedin Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168,which is incorporated by reference. A wide variety of equipment iscommercially available for automated oligonucleotide synthesis.Multi-nucleotide synthesis approaches (e.g., tri-nucleotide synthesis,etc.) are also optionally utilized. Moreover, the primer nucleic acidsoptionally include various modifications. In certain embodiments, forexample, primers include restriction site linkers, e.g., to facilitatesubsequent amplicon cloning or the like. To further illustrate, primersare also optionally modified to improve the specificity of amplificationreactions as described in, e.g., U.S. Pat. No. 6,001,611, entitled“MODIFIED NUCLEIC ACID AMPLIFICATION PRIMERS,” issued Dec. 14, 1999 toWill, which is incorporated by reference. Primers and probes can also besynthesized with various other modifications (e.g., restriction sites,enzyme binding sites, etc.) as described herein or as otherwise known inthe art.

Probes and/or primers utilized in the reaction mixtures, methods, andother aspects of the invention are typically labeled to permit detectionof probe-target hybridization duplexes. In general, a label can be anymoiety that can be attached to a nucleic acid and provide a detectablesignal (e.g., a quantifiable signal). Labels may be attached tooligonucleotides directly or indirectly by a variety of techniques knownin the art. To illustrate, depending on the type of label used, thelabel can be attached to a terminal (5′ or 3′ end of an oligonucleotideprimer and/or probe) or a non-terminal nucleotide, and can be attachedindirectly through linkers or spacer arms of various sizes andcompositions. Using commercially available phosphoramidite reagents, onecan produce oligonucleotides containing functional groups (e.g., thiolsor primary amines) at either the 5′ or 3′ terminus via an appropriatelyprotected phosphoramidite, and can label such oligonucleotides usingprotocols described in, e.g., Innis et al. (Eds.) PCR Protocols: A Guideto Methods and Applications, Elsevier Science & Technology Books(1990)(Innis), which is incorporated by reference.

Essentially any labeling moiety is optionally utilized to label a probeand/or primer by techniques well known in the art. In some embodiments,for example, labels comprise a fluorescent dye (e.g., a rhodamine dye(e.g., R6G, R110, TAMRA, ROX, etc.), a fluorescein dye (e.g., JOE, VIC,TET, HEX, FAM, etc.), a halofluorescein dye, a cyanine dye (e.g., CY3,CY3.5, CY5, CY5.5, etc.), a BODIPY® dye (e.g., FL, 530/550, TR, TMR,etc.), an ALEXA FLUOR® dye (e.g., 488, 532, 546, 568, 594, 555, 653,647, 660, 680, etc.), a dichlororhodamine dye, an energy transfer dye(e.g., BIGDYE™ v 1 dyes, BIGDYE™ v 2 dyes, BIGDYE™ v 3 dyes, etc.),Lucifer dyes (e.g., Lucifer yellow, etc.), CASCADE BLUE®, Oregon Green,and the like. Additional examples of fluorescent dyes are provided in,e.g., Haugland, Molecular Probes Handbook of Fluorescent Probes andResearch Products, Ninth Ed. (2003) and the updates thereto, which areeach incorporated by reference. Fluorescent dyes are generally readilyavailable from various commercial suppliers including, e.g., MolecularProbes, Inc. (Eugene, Oreg.), Armersham Biosciences Corp. (Piscataway,N.J.), Applied Biosystems (Foster City, Calif.), etc. Other labelsinclude, e.g., biotin, weakly fluorescent labels (Yin et al. (2003) ApplEnviron Microbiol. 69(7):3938, Babendure et al. (2003) Anal. Biochem.317(1):1, and Jankowiak et al. (2003) Chem Res Toxicol. 16(3):304),non-fluorescent labels, colorimetric labels, chemiluminescent labels(Wilson et al. (2003) Analyst. 128(5):480 and Roda et al. (2003)Luminescence 18(2):72), Raman labels, electrochemical labels,bioluminescent labels (Kitayama et al. (2003) Photochem Photobiol.77(3):333, Arakawa et al. (2003) Anal. Biochem. 314(2):206, and Maeda(2003) J. Pharm. Biomed. Anal. 30(6): 1725), and an alpha-methyl-PEGlabeling reagent as described in, e.g., U.S. Provisional PatentApplication No. 60/428,484, filed on Nov. 22, 2002, which references areeach incorporated by reference. Nucleic acid labeling is also describedfurther below.

In addition, whether a fluorescent dye is a donor or an acceptor isgenerally defined by its excitation and emission spectra, and thefluorescent dye with which it is paired. Fluorescent molecules commonlyused as quencher moieties in probes and primers include, e.g.,fluorescein, FAM, JOE, rhodamine, R6G, TAMRA, ROX, DABCYL, and EDANS.Many of these compounds are available from the commercial suppliersreferred to above. Exemplary non-fluorescent or dark quenchers thatdissipate energy absorbed from a fluorescent dye include the Black HoleQuenchers™ or BHQ™, which are commercially available from BiosearchTechnologies, Inc. (Novato, Calif., USA).

To further illustrate, essentially any nucleic acid (and virtually anylabeled nucleic acid, whether standard or non-standard) can be custom orstandard ordered from any of a variety of commercial sources, such asThe Midland Certified Reagent Company, The Great American Gene Company,ExpressGen Inc., Operon Technologies Inc., Proligo LLC, and many others.

In certain embodiments, modified nucleotides are included in probes andprimers. To illustrate, the introduction of modified nucleotidesubstitutions into oligonucleotide sequences can, e.g., increase themelting temperature of the oligonucleotides. In some embodiments, thiscan yield greater sensitivity relative to corresponding unmodifiedoligonucleotides even in the presence of one or more mismatches insequence between the target nucleic acid and the particularoligonucleotide. Exemplary modified nucleotides that can be substitutedor added in oligonucleotides include, e.g., C5-ethyl-dC, C5-ethyl-dU,2,6-diaminopurines, C5-propynyl-dC, C7-propynyl-dA, C7-propynyl-dG,C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA,C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidineanalogs, pseudo-dU, nitro pyrrole, nitro indole, 2′-0-methyl Ribo-U,2′-0-methyl Ribo-C, an 8-aza-dA, an 8-aza-dG, a 7-deaza-dA, a7-deaza-dG, N4-ethyl-dC, N6-methyl-dA, etc. To further illustrate, otherexamples of modified oligonucleotides include those having one or moreLNA™ monomers. Nucleotide analogs such as these are also described in,e.g., U.S. Pat. No. 6,639,059, entitled “SYNTHESIS OF [2.2.1]BICYCLONUCLEOSIDES,” issued Oct. 28, 2003 to Kochkine et al., U.S. Pat. No.6,303,315, entitled “ONE STEP SAMPLE PREPARATION AND DETECTION OFNUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES,” issued Oct. 16, 2001 toSkouv, and U.S. Pat. Application Pub. No. 2003/0092905, entitled“SYNTHESIS OF [2.2.1]BICYCLO NUCLEOSIDES,” by Kochkine et al. thatpublished May 15, 2003, which are each incorporated by reference.Oligonucleotides comprising LNA™ monomers are commercially availablethrough, e.g., Exiqon A/S (Vedbaek, DK). Additional oligonucleotidemodifications are referred to herein, including in the definitionsprovided above.

Labeled oligonucleotides, such as 5′-nuclease probes, hybridizationprobes, SCORPION® primers, and molecular beacons are described furtherherein.

Nucleic Acid Amplification Reagents

The reaction mixtures of the invention typically include selectedamounts of light emission modifiers and labeled oligonucleotides, asdescribed herein. In addition, reaction mixtures also generally includevarious reagents that are useful in performing nucleic acidamplification or detection reactions, such as real-time PCR monitoringor 5′-nuclease assays. Exemplary nucleic acid amplification reagentsinclude, e.g., primer nucleic acids, template or target nucleic acids,nucleotide incorporating biocatalysts (e.g., DNA polymerases, etc.),extendible nucleotides, terminator nucleotides, buffers, salts,amplicons, glycerol, metal ions, dimethyl sulfoxide (DMSO), poly rA (acarrier nucleic acid for low copy targets), and the like. In someembodiments, for example, nucleic acid amplification reactions areperformed utilizing these reaction mixtures to effect the detection oftarget nucleic acids in samples, e.g., to aid in the diagnosis and/orprognosis of diseases. Nucleic acid amplification and detection methodsare also described further below.

Reaction mixtures are generally produced by combining selected lightemission modifiers and labeled oligonucleotides with quantities of thenucleic acid amplification reagents that are sufficient for performingthe particular nucleic acid amplification method selected. Thequantities of nucleic acid amplification reagents to be included in agiven reaction mixture are well-known to persons of skill in the art inview of the selected nucleic acid amplification method. To illustrate,however, primer nucleic acids and extendible nucleotides (e.g., fourdNTPs (dGTP, dCTP, dATP, dTTP)) are each present in a large molar excessin the reaction mixtures in certain embodiments. Probe and primernucleic acids that can be utilized in the reaction mixtures of theinvention are described herein. Suitable extendible nucleotides arereadily available from many different commercial suppliers including,e.g., Roche Diagnostics Corporation (Indianapolis, Ind., USA), AmershamBiosciences Corp. (Piscataway, N.J., USA), Applied Biosystems (FosterCity, Calif., USA), and the like.

The nucleotide incorporating biocatalysts utilized in the reactionmixtures and other aspect of the invention typically comprise enzymes,such as polymerases, terminal transferases, reverse transcriptases,telomerases, polynucleotide phosphorylases, and the like. In certainembodiments, for example, the enzyme includes a 5′-3′ nuclease activity,a 3′-5′ exonuclease activity, and/or is a thermostable enzyme. Theenzyme is optionally derived from an organism, such as Thermusantranikianii, Thermus aquaticus, Thermus caldophilus, Thermuschliarophilus, Thermusfiliformis, Thermus flavus, Thermus igniterrae,Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermusscotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana,Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax,Bacillus stearothermophilus, or the like.

In certain embodiments, additional reagents are also added to thereaction mixtures of the invention. To illustrate, reaction mixturesalso optionally include pyrophosphatases (e.g., a thermostablepyrophosphatase), e.g., for use in minimizing pyrophosphorolysis, dUTPand uracil N-glycosylase (UNG) (e.g., a thermostable UNG), e.g., toprotect against carry-over contamination, and the like.

Methods of Modifying Light Emissions from Labeled Oligonucleotides

The invention also provides methods of modifying light emissions (e.g.,baseline light emissions) from labeled oligonucleotides. Typically,these methods are performed as part of assays that involve the detectionof target nucleic acids, e.g., to provide diagnostic, genetic, or otherinformation about subjects from which the target nucleic acids werederived. In some embodiments, the light emission modifiers used in thesemethods reduce the emission of light from labeled oligonucleotides. Thisgenerally improves performance characteristics, such as the sensitivityand dynamic range of the particular assay (e.g., a real-time PCRtechnique) in which the light emission modifiers described herein areutilized. These aspects are also illustrated in the examples providedbelow.

In practicing the methods of the present invention, many conventionaltechniques in molecular biology are optionally utilized. Thesetechniques are well known and are explained in, for example, Ausubel etal. (Eds.) Current Protocols in Molecular Biology, Volumes I, II, andIII, (1997) (Ausubel 1), Ausubel et al. (Eds.), Short Protocols inMolecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, 5^(th) Ed., John Wiley & Sons, Inc. (2002) (Ausubel2), Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed.,Cold Spring Harbor Laboratory Press (2000) (Sambrook), Berger andKimmel, Guide to Molecular Cloning Techniques: Methods in Enzymology,Volume 152, Academic Press, Inc. (Berger), Vorbruggen et al., Handbookof Nucleoside Synthesis, Organic Reactions Series, #60, John Wiley &Sons, Inc. (2001), Gait (Ed.) Oligonucleotide Synthesis, OxfordUniversity Press (1984), Hames and Higgins, Nucleic Acid Hybridization,Practical Approach Series, Oxford University Press (1997), and Hames andHiggins (Eds.) Transcription and Translation, Practical Approach Series,Oxford University Press (1984), all of which are incorporated byreference.

Examples of general types of nucleic acid analysis technologies that canbe used or adapted for use to analyze target nucleic acids in or fromthe reactions mixtures of the invention include various nucleic acidamplification assays. A common characteristic among nucleic acidamplification assays is that they are typically designed to amplifynucleic acid sequences that are specific for the organism beingdetected. Nucleic acid amplification tests generally have greatersensitivity than other approaches to nucleic acid analysis. Thissensitivity, which is further improved with the use of the lightemission modifiers of the invention, is typically attributable to theirability to produce a positive signal from as little as a single copy ofthe target nucleic acid. Amplification methods that are optionallyutilized or adapted to detect target nucleic acids include, e.g.,various polymerase, ligase, or reverse-transcriptase mediatedamplification methods, such as the polymerase chain reaction (PCR), theligase chain reaction (LCR), reverse-transcription PCR(RT-PCR), NASBA,TMA, SDA and the like. Additional details regarding the use of these andother amplification methods and various approaches to sample preparationfor these assays can be found in any of a variety of standard texts,including, e.g., Berger, Sambrook, Ausubel 1 and 2, and Innis, which arereferred to above. Various commercial nucleic acid amplification assaysthat are optionally adapted for use with the light emission modifiersand methods of the invention generally differ in their amplificationmethods and their target nucleic acid sequences. Examples of thesecommercial tests include the AMPLICOR® and COBAS AMPLICOR® assays (RocheDiagnostics Corporation, Indianapolis, Ind., USA), which use polymerasechain reactions (PCR); the LCx® test (Abbott Laboratories, Abbott Park,Ill., USA), which uses ligase chain reactions (LCR); the BDProbeTec™ ETtest (Becton, Dickinson and Company, Franklin Lakes, N.J., USA), whichuses strand displacement amplification (SDA); the NucliSens EasyQ assay(bioMerieux, Durham, N.C.), which uses nucleic acid sequence-basedamplification (NASBA); and the APTIMA™ assay (Gen-Probe, Inc., SanDiego, Calif., USA), which uses transcription-mediated amplification(TMA). Nucleic acid amplification and detection is described furtherbelow.

In certain embodiments, for example, the light emission modifiers of theinvention are utilized in various 5′-nuclease reactions to modify (e.g.,reduce) light emissions from 5′-nuclease probes. Many 5′-nuclease assaysare well known to those of skill in the art. Examples of such reactionsare also described, for instance, in U.S. Pat. Nos. 5,210,015,6,214,979, 5,804,375, and 5,487,972, supra, which are each incorporatedby reference.

To briefly illustrate, in a 5′-nuclease reaction, a target nucleic acidis contacted with a primer and a probe (e.g., 5′-nuclease probe, etc.)under conditions in which the primer and probe hybridize to a strand ofthe target nucleic acid. The target nucleic acid, primer and probe arealso contacted with a selected amount of a light emission modifier and anucleic acid polymerase having 5′ to 3′ nuclease activity. Nucleic acidpolymerases possessing 5′ to 3′ nuclease activity can cleave the probehybridized to the target nucleic acid downstream of the primer. The 3′end of the primer provides the initial binding site for the polymerase.The bound polymerase cleaves fragments from the probe upon encounteringthe 5′ end of the probe.

The primer and probe can be designed such that they anneal in closeproximity on the target nucleic acid such that binding of the nucleicacid polymerase to the 3′ end of the primer puts it in contact with the5′ end of the probe in the absence of primer extension. The term“polymerization-independent cleavage” refers to this process.Alternatively, if the primer and probe anneal to more distantly spacedregions of the target nucleic acid, polymerization typically occursbefore the nucleic acid polymerase encounters the 5′ end of the probe.As the polymerization continues, the polymerase progressively cleavesfragments from the 5′ end of the probe. This cleaving continues untilthe remainder of the probe has been destabilized to the extent that itdissociates from the template molecule. The term“polymerization-dependent cleavage” refers to this process.

One advantage of polymerization-independent cleavage lies in theelimination of the need for amplification of the nucleic acid. Providedthe primer and probe are adjacently bound to the nucleic acid,sequential rounds of probe annealing and cleavage of fragments canoccur. Thus, a sufficient amount of fragments can be generated, makingdetection possible in the absence of polymerization.

In either process, a sample is provided which is thought to contain thetarget nucleic acid. The target nucleic acid contained in the sample maybe first reverse transcribed into cDNA, if necessary, and thendenatured, using any suitable denaturing method, including physical,chemical, or enzymatic methods, which are known to those of skill in theart. An exemplary physical approach to effect strand separation involvesheating the nucleic acid until it is completely (>99%) denatured.Typical heat denaturation involves temperatures ranging from about 85°C. to about 105° C., for periods of time ranging from about 1 to about10 minutes. As an alternative to denaturation, the nucleic acid mayexist in a single-stranded form in the sample, such as, for example,single stranded RNA or DNA viruses.

The denatured target nucleic acid strand is typically incubated with aprimer, a probe, and the selected light emission modifier underhybridization conditions that permit the primer and probe to bind to thetarget nucleic acid strand and the light emission modifier to bind atleast to the probe. In some embodiments, two primers can be used toamplify the target nucleic acid. The two primers are typically selectedso that their relative positions along the target nucleic acid are suchthat an extension product synthesized from one primer, when theextension produce is separated from its template (complement), serves asa template for the extension of the other primer to yield a replicatestrand of defined length. In multiplexing formats, multiple probes aretypically used in a single reaction vessel to simultaneously detectmultiple target nucleic acids.

Because the complementary strands are typically longer than either theprobe or primer, the strands have more points of contact and thus agreater chance of binding to each other over a given period of time.Accordingly, a high molar excess of probe and primer is typicallyutilized to favor primer and probe annealing over template strandreannealing.

Primers are generally of sufficient length and complementarity so thatthey selectively bind to target nucleic acids under selected conditionsto permit polymerization-independent cleavage orpolymerization-dependent cleavage to proceed. The exact length andcomposition of the primer will depend on many factors, includingtemperature of the annealing reaction, source and composition of theprimer, proximity of the probe annealing site to the primer annealingsite, and ratio of primer:probe concentration. For example, depending onthe complexity of the target sequence, the primer typically includesabout 15-30 nucleotides, although it may contain more or fewernucleotides.

The probe is generally annealed to its complementary target nucleic acidbefore the nucleic acid polymerase encounters that region of the targetnucleic acid, thereby permitting the 5′ to 3′ nuclease activity of theenzyme to cleave fragments from the probe. To enhance the likelihoodthat the probe will anneal to the target nucleic acid before thepolymerase reaches this region of hybridization, a variety of techniquesmay be utilized. For example, short primers generally require coolertemperature to form sufficiently stable hybrid complexes with thenucleic acid. Therefore, the probe can be designed to be longer than theprimer so that the probe preferentially anneals to the target nucleicacid at higher temperatures relative to primer annealing. To furtherillustrate, primers and probes having differential thermal stability canalso be utilized. For example, the nucleotide composition of the probecan be chosen to have greater G/C content and, consequently, greaterthermal stability than the primer. Optionally, modified nucleotides maybe incorporated into primers or probes to effect either greater orlesser thermal stability in comparison to primers or probes having onlyunmodified nucleotides. Exemplary modified nucleotides are describedfurther above. The thermocycling parameters can also be varied to takeadvantage of the differential thermal stability of the probe and primer.For example, following a thermocycling denaturation step, anintermediate temperature may be introduced which permits probe bindingbut not primer binding. Thereafter, the temperature can be furtherreduced to permit primer annealing. To preferentially favor binding ofthe probe before the primer, a high molar excess of probe to primerconcentration can also be used. Such probe concentrations are typicallyin the range of about 2 to about 20 times higher than the respectiveprimer concentration, which is generally about 0.5-5×10⁻⁷ M.

Template-dependent extension of primers is generally catalyzed by anucleotide incorporating biocatalyst (e.g., a polymerase, etc.) in thepresence of adequate amounts of the four deoxyribonucleosidetriphosphates (dATP, dGTP, dCTP, and dTTP) or analogs in a reactionmixture that also includes light emission modifiers and appropriatesalts, metal cations, and buffers. Reaction mixtures are describedfurther above. Suitable nucleotide incorporating biocatalysts areenzymes known to catalyze primer and template-dependent DNA synthesisand possess the 5′ to 3′ nuclease activity. Exemplary DNA polymerases ofthis type include E. coli DNA polymerase I, Tth DNA polymerase, Bacillusstearothermophilus DNA polymerase, Taq DNA polymerase, Thermus sp. ZO5DNA polymerase, Thermatoga maritima DNA polymerase, Thermatoganeopolitana DNA polymerase, and Thermosipho africanus DNA polymerase.The reaction conditions for catalyzing DNA synthesis with these DNApolymerases are well known in the art. Typically, the nucleotideincorporating biocatalyst efficiently cleaves the probe and releaseslabeled fragments so that a detectable signal is directly or indirectlygenerated.

The products of the synthesis are generally duplex molecules thatinclude the template strands and the primer extension strands.Byproducts of this synthesis are probe fragments, which can include amixture of mono-, di- and larger nucleotide fragments. Repeated cyclesof denaturation, probe and primer annealing, and primer extension andprobe cleavage result in the exponential accumulation of the regiondefined by the primers and the exponential generation of labeledfragments. Sufficient cycles are run to achieve a detectable amount ofprobe fragments, which is generally several orders of magnitude greaterthan background signal. The use of light emission modifiers as describedherein can effectively reduce the number of cycles run before adetectable signal is produced relative to assays that do not reducethese background signals.

In certain embodiments, PCR reactions are carried out as an automatedprocess, which utilizes a thermostable enzyme. In this process thereaction mixture is cycled through a denaturing step, a probe and primerannealing step, and a synthesis step in which cleavage and displacementoccur simultaneously with primer dependent template extension. In someembodiments, the methods described herein are performed using a system.Such systems are described in greater detail below. Optionally, thermalcyclers, such as those commercially available from, e.g., AppliedBiosystems (Foster City, Calif., USA), which are designed for use withthermostable enzymes, may be utilized.

Thermostable polymerases are typically used in automated processes thateffect the denaturation of double stranded extension products byexposing them to a elevated temperatures (e.g., about 95° C.) during thePCR cycle. For example, U.S. Pat. No. 4,889,818, entitled “PURIFIEDTHERMOSTABLE ENZYME,” issued to Dec. 26, 1989 to Gelfand et al., whichis incorporated by reference, discloses a representative thermostableenzyme isolated from Thermus aquaticus. Additional representativethermostable polymerases include, e.g., polymerases extracted from thethermostable bacteria Thermus flavus, Thermus ruber, Thermusthermophilus, Bacillus stearothermophilus (which has a somewhat lowertemperature optimum than the others listed), Thermus lacteus, Thermusrubens, Thermotoga maritima, Thermatoga neopolitana, Thermosiphoafricanus, Thermococcus littoralis, and Methanothermus fervidus.

Hybridization of probes to target nucleic acids can be accomplished bychoosing appropriate hybridization conditions. The stability of theprobe:target nucleic acid hybrid is typically selected to be compatiblewith the assay and washing conditions so that stable, detectable hybridsform only between the probes and target nucleic acids. Manipulation ofone or more of the different assay parameters determines the exactsensitivity and specificity of a particular hybridization assay.

More specifically, hybridization between complementary bases of DNA,RNA, PNA, or combinations of DNA, RNA and PNA, occurs under a widevariety of conditions that vary in temperature, salt concentration,electrostatic strength, buffer composition, and the like. Examples ofthese conditions and methods for applying them are described in, e.g.,Tijssen, Hybridization with Nucleic Acid Probes, Vol. 24, ElsevierScience (1993), and Hames and Higgins, supra, which are bothincorporated by reference. Hybridization generally takes place betweenabout 0° C. and about 70° C., for periods of from about one minute toabout one hour, depending on the nature of the sequence to be hybridizedand its length. However, it is recognized that hybridizations can occurin seconds or hours, depending on the conditions of the reaction. Toillustrate, typical hybridization conditions for a mixture of two20-mers is to bring the mixture to 68° C., followed by cooling to roomtemperature (22° C.) for five minutes or at very low temperatures suchas 2° C. in 2 microliters. Hybridization between nucleic acids may befacilitated using buffers such as Tris-EDTA (TE), Tris-HCl and HEPES,salt solutions (e.g. NaCl, KCl, CaCl₂), or other aqueous solutions,reagents and chemicals. Examples of these reagents includesingle-stranded binding proteins such as Rec A protein, T4 gene 32protein, E. coli single-stranded binding protein and major or minornucleic acid groove binding proteins. Other examples of such reagentsand chemicals include divalent ions, polyvalent ions and intercalatingsubstances such as ethidium bromide, actinomycin D, psoralen, andangelicin.

Essentially any available method for detecting target nucleic acids canbe used in the present invention. Common approaches include real-timeamplification detection with 5′-nuclease probes, SCORPION® primers, ormolecular beacons, detection of intercalating dyes, detection of labelsincorporated into the amplification probes or the amplified nucleicacids themselves, e.g., following electrophoretic separation of theamplification products from unincorporated label, hybridization basedassays (e.g., array based assays), and/or detection of secondaryreagents that bind to the nucleic acids. For example, a 5′-nucleaseprobe or a molecular beacon is optionally designed to include aoligonucleotide sequence that targets a particular nucleic acid (e.g., anucleic acid from Neisseria gonorrhoeae, Neisseria meningitidis, humanimmunodeficiency virus (HIV), hepatitis C virus (HCV), papilloma virus,Plasmodium falciparum, Chlamydia muridarum, Chlamydia trachomatis, amongmany others). Molecular beacons and 5′-nuclease probes are describedfurther below. These general approaches are also described in, e.g.,Sambrook, and Ausubel 1 and 2.

In certain embodiments, real-time PCR assay systems that include one ormore 5′-nuclease probes are used for detecting amplified target nucleicacids in the presence of the light emission modifiers described herein.As described above, these systems operate by using the endogenousnuclease activity of certain polymerases to cleave a quencher or otherlabel free from a probe that comprises the quencher and label, resultingin unquenching of the label. The polymerase typically only cleaves thequencher or label upon initiation of replication, i.e., when theoligonucleotide is bound to the template and the polymerase extends theprimer. Thus, an appropriately labeled probe nucleic acid and apolymerase comprising the appropriate nuclease activity can be used todetect a target nucleic acid of interest. Real-time PCR product analysisby, e.g., FRET or the like (and related kinetic reverse-transcriptionPCR) provides a well-known technique for real time PCR monitoring thathas been used in a variety of contexts, which can be adapted for usewith the methods described herein (see, Laurendeau et al. (1999) “TaqManPCR-based gene dosage assay for predictive testing in individuals from acancer family with INK4 locus haploinsufficiency” Clin Chem 45(7):982-6;Laurendeau et al. (1999) “Quantitation of MYC gene expression insporadic breast tumors with a real-time reverse transcription-PCR assay”Clin Chem 59(12):2759-65; and Kreuzer et al. (1999) “LightCyclertechnology for the quantitation of bcr/ab1 fusion transcripts” CancerResearch 59(13):3171-4, all of which are incorporated by reference).

Exemplary commercially available systems that are optionally utilized todetect target nucleic acids using the reaction mixtures described hereininclude, e.g., a COBAS® TaqMan® system, a COBAS AMPLICOR® Analyzer, or aLightCycler® system, which are available from Roche DiagnosticsCorporation (Indianapolis, Ind., USA), a LUMINEX 100™ system, which isavailable from the Luminex Corporation (Austin, Tex., USA), a ABI PRISM®7700 system, which is available from Applied Biosystems (Foster City,Calif., USA), and the like. Systems are also described below.

Molecular beacons are oligonucleotides designed for real-time detectionand quantification of target nucleic acids. The 5′ and 3′ termini ofmolecular beacons collectively comprise a pair of moieties, whichconfers the detectable properties of the molecular beacon. One of thetermini is attached to a fluorophore and the other is attached to aquencher molecule capable of quenching a fluorescent emission of thefluorophore. To illustrate, one example fluorophore-quencher pair canuse a fluorophore, such as EDANS or fluorescein, e.g., on the 5′-end anda quencher, such as Dabcyl, e.g., on the 3′-end. When the molecularbeacon is present free in solution, i.e., not hybridized to a secondnucleic acid, the stem of the molecular beacon is stabilized bycomplementary base pairing. This self-complementary pairing results in a“hairpin loop” structure for the molecular beacon in which thefluorophore and the quenching moieties are proximal to one another. Inthis confirmation, the fluorescent moiety is quenched by the quenchingmoiety. The loop of the molecular beacon typically comprises theoligonucleotide probe and is accordingly complementary to a sequence tobe detected in the target nucleic acid, such that hybridization of theloop to its complementary sequence in the target forces disassociationof the stem, thereby distancing the fluorophore and quencher from eachother. This results in unquenching of the fluorophore, causing anincrease in fluorescence of the molecular beacon.

Details regarding standard methods of making and using molecular beaconsare well established in the literature and molecular beacons areavailable from a number of commercial reagent sources. Further detailsregarding methods of molecular beacon manufacture and use are found,e.g., in Leone et al. (1995) “Molecular beacon probes combined withamplification by NASBA enable homogenous real-time detection of RNA,”Nucleic Acids Res. 26:2150-2155; Kostrikis et al. (1998) “Molecularbeacons: spectral genotyping of human alleles” Science 279:1228-1229;Fang et al. (1999) “Designing a novel molecular beacon forsurface-immobilized DNA hybridization studies” J. Am. Chem. Soc.121:2921-2922; and Marras et al. (1999) “Multiplex detection ofsingle-nucleotide variation using molecular beacons” Genet. Anal.Biomol. Eng. 14:151-156, all of which are incorporated by reference. Avariety of commercial suppliers produce standard and custom molecularbeacons, including Oswel Research Products Ltd. (UK), Research Genetics(a division of Invitrogen, Huntsville, Ala., USA), the Midland CertifiedReagent Company (Midland, Tex., USA), and Gorilla Genomics, LLC(Alameda, Calif., USA). A variety of kits which utilize molecularbeacons are also commercially available, such as the Sentinel™ MolecularBeacon Allelic Discrimination Kits from Stratagene (La Jolla, Calif.,USA) and various kits from Eurogentec SA (Belgium) and Isogen BioscienceBV (Netherlands).

SCORPION® primers are used in fluorescence based approaches for thespecific detection of PCR products (Whitcombe et al. (1999) Nat.Biotechnol. 17:804-807, Whitcome et al. (1999) Am J. Hum. Genet.65:2333, and Thelwell et al. (2000) Nucl. Acids Res. 28:3752-3761, whichare each incorporated by reference). A SCORPION® primer generallyincludes a specific probe sequence that is held in a hairpin loopconfiguration by complementary stem sequences on the 5′ and 3′ sides ofthe probe. The fluorescent label attached to the 5′-end is quenched by aquencher moiety attached to the 3′-end of the loop. The hairpin loop islinked to the 5′-end of a primer typically via a PCR stopper. Afterextension of the primer during PCR amplification, the specific probesequence is able to bind to its complement within the same strand ofDNA. This hybridization event opens the hairpin loop so thatfluorescence is no longer quenched and an increase in signal isobserved. The PCR stopper prevents read-through, which can lead toopening the hairpin loop in the absence of the specific target sequence.Such read-through would lead to the detection of non-specific PCRproducts, such as primer dimers or mispriming events. SCORPION® primersare also described in, e.g., Huang et al. (2004) “Real-time quantitativeassay of telomerase activity using the duplex scorpion primer,”Biotechnol Lett. 26(11):891-895, Asselbergs et al. (2003) “Rapiddetection of apoptosis through real-time reverse transcriptasepolymerase chain reaction measurement of the small cytoplasmic RNA Y1,”Anal Biochem. 318(2):221-229, and Nuovo et al. (1999) “In situamplification using universal energy transfer-labeled primers,” JHistochem Cytochem. 47(3):273-280, which are each incorporated byreference.

Systems

The invention also provides systems for detecting target nucleic acids.The system includes one or more labeled oligonucleotides and one or morelight emission modifiers as described herein. In certain embodiments,the oligonucleotides are arrayed on a solid support, whereas in others,they are provided in one or more containers, e.g., for assays performedin solution. The system also includes at least one detector (e.g., aspectrometer, etc.) that detects binding between nucleic acids and/oramplicons thereof from the sample and the oligonucleotides. In addition,the systems also optionally include at least one thermal modulator(e.g., a thermal cycling device, etc.) operably connected to thecontainer or solid support to modulate temperature in the container oron the solid support, and/or at least one fluid transfer component(e.g., an automated pipettor, etc.) that transfers fluid to and/or fromthe container or solid support, e.g., for performing one or more nucleicacid amplification techniques and/or nucleic acid hybridization assaysin the container or on the solid support.

Detectors are typically structured to detect detectable signalsproduced, e.g., in or proximal to another component of the given assaysystem (e.g., in container, on a solid support, etc.). Suitable signaldetectors that are optionally utilized, or adapted for use, hereindetect, e.g., fluorescence, phosphorescence, radioactivity, absorbance,refractive index, luminescence, mass, or the like. Detectors optionallymonitor one or a plurality of signals from upstream and/or downstream ofthe performance of, e.g., a given assay step. For example, detectorsoptionally monitor a plurality of optical signals, which correspond inposition to “real-time” results. Example detectors or sensors includephotomultiplier tubes, CCD arrays, optical sensors, temperature sensors,pressure sensors, pH sensors, conductivity sensors, scanning detectors,or the like. More specific exemplary detectors that are optionallyutilized in performing the methods of the invention include, e.g.,resonance light scattering detectors, emission spectroscopes,fluorescence spectroscopes, phosphorescence spectroscopes, luminescencespectroscopes, spectrophotometers, photometers, and the like. Detectorsare also described in, e.g., Skoog et al., Principles of InstrumentalAnalysis, 5^(th) Ed., Harcourt Brace College Publishers (1998) andCurrell, Analytical Instrumentation: Performance Characteristics andQuality, John Wiley & Sons, Inc. (2000), both of which are incorporatedby reference.

The systems of the invention also typically include controllers that areoperably connected to one or more components (e.g., detectors, thermalmodulators, fluid transfer components, etc.) of the system to controloperation of the components. More specifically, controllers aregenerally included either as separate or integral system components thatare utilized, e.g., to receive data from detectors, to effect and/orregulate temperature in the containers, to effect and/or regulate fluidflow to or from selected containers, or the like. Controllers and/orother system components is/are optionally coupled to an appropriatelyprogrammed processor, computer, digital device, or other informationappliance (e.g., including an analog to digital or digital to analogconverter as needed), which functions to instruct the operation of theseinstruments in accordance with preprogrammed or user input instructions,receive data and information from these instruments, and interpret,manipulate and report this information to the user. Suitable controllersare generally known in the art and are available from various commercialsources.

Any controller or computer optionally includes a monitor, which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display, etc.), or others.Computer circuitry is often placed in a box, which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser. These components are illustrated further below.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set of parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of one or more controllers to carry out thedesired operation. The computer then receives the data from, e.g.,sensors/detectors included within the system, and interprets the data,either provides it in a user understood format, or uses that data toinitiate further controller instructions, in accordance with theprogramming, e.g., such as controlling fluid flow regulators in responseto fluid weight data received from weight scales or the like.

FIG. 3 is a schematic showing a representative system that includes alogic device in which various aspects of the present invention may beembodied. As will be understood by practitioners in the art from theteachings provided herein, the invention is optionally implemented inhardware and/or software. In some embodiments, different aspects of theinvention are implemented in either client-side logic or server-sidelogic. As will be understood in the art, the invention or componentsthereof may be embodied in a media program component (e.g., a fixedmedia component) containing logic instructions and/or data that, whenloaded into an appropriately configured computing device, cause thatdevice to perform according to the invention. As will also be understoodin the art, a fixed media containing logic instructions may be deliveredto a viewer on a fixed media for physically loading into a viewer'scomputer or a fixed media containing logic instructions may reside on aremote server that a viewer accesses through a communication medium inorder to download a program component.

In particular, FIG. 3 schematically illustrates computer 300 to whichdetector 302, fluid transfer component 304, and thermal modulator 308are operably connected. Optionally, one or more of these components areoperably connected to computer 300 via a server (not shown in FIG. 3).During operation, fluid transfer component 304 typically transfersreaction mixtures or components thereof to multi-well container 306.Thermal cycling is typically effected by thermal modulator 308, whichthermally communicates with multi-well container 306. Detector 302typically detects detectable signals (e.g., fluorescent emissions, etc.)produced during thermal cycling reactions.

Kits

The reaction mixtures or components thereof (e.g., probes or lightemission modifiers) employed in the methods of the present invention areoptionally packaged into kits. In addition, the kits may also includesuitably packaged reagents and materials needed for target nucleic acidhybridization, amplification, and detection, such buffers, enzymes, DNAstandards, salts, metal ions, primers, extendible or terminatornucleotides, glycerol, dimethyl sulfoxide, poly rA, etc. as well asinstructions for conducting a particular assay. Kit components, such asprobes and light emission modifiers are typically provided in one ormore containers. In some of these embodiments, the kits further includeat least one pyrophosphatase (e.g., a thermostable pyrophosphatase),e.g., for use in minimizing pyrophosphorolysis, and/or uracilN-glycosylase (UNG), e.g., for use in applications where protectionagainst carry-over contamination is desirable. Two or more of the kitcomponents may be packaged within the same container.

The Use of Soluble Light Emission Modifiers in Tm Determinations

The invention provides methods for determining the melting temperature(T_(m)) of a hybridization complex, where these methods use the solublelight emission modifier technology taught herein. The Tm determinationsuse a soluble light emission modifier (i.e., a soluble quencher) systemto monitor the duplex melting curve or annealing curve.

Essentially, a probe labeled with a suitable light emitting moiety(e.g., a donor) is hybridized with a target to form a hybridizationcomplex. The resulting hybridization duplex (e.g., target hybridizationcomplex) can have either complete complementarity (i.e., 100%) orpartial complementarity (i.e., less than 100%). Any nucleic acid duplex(with complete or partial complementarity) is characterized by aparticular Tm at a give set of hybridization conditions. It is thisfeature that makes Tm determinations useful in applications such asdiagnostics (e.g., SNP detection, mutation detection and mutationscanning, viral genotyping, testing for drug resistant strains etc.).

Either before, during or after formation of the duplex, the reaction isadmixed with a suitable soluble quencher. This soluble quenchercomprises a thiazine dye or a diazine dye, where the soluble quencher iscapable of quenching the light emitting moiety on the probe (thusforming a donor-acceptor pair). Any thiazine dye or diazine dye providedherein can be used in the Tm determination methods of the invention. Itis noted that thiazine, phenothiazine, cationic thiazines, thiazinium,and phenothiazinium are all synonyms for a generic name for the familyof dyes with fused 3-ring aromatic system containing a nitrogen and asulfur in the middle ring. Furthermore, in addition to the particularthiazine and diazine structures taught herein, related structuralvariants of these molecules that retain the soluble quencher propertycan also be used with the methods of the invention, and are encompassedwithin the scope of the invention.

A thiazine dye or diazine dye soluble quencher acts by binding to bothsingle and double-stranded nucleic acid, but has reduced affinity forsingle-stranded nucleic acid. It is contemplated that the binding to thesingle stranded nucleic acids could be due to partial secondarystructures in the random coil state. Without being bound to anyparticular theory, it is believed that the predominant binding mode isthrough intercalation, but minor and major groove binding is alsopossible depending on the sequence context and hybridization conditions(see, Rohs et al. (2000) J. Am. Chem. Soc., 122:2860-2866; and Tuite etal. (1994) J. Am. Chem. Soc., 116:7548-7556). Thus, the fluorescencedonor label attached to the probe that forms the hybridization complexwith a target polynucleotide is subject to a quenching effect by theintercalating soluble quencher that has an affinity for double-strandednucleic acid due to the close proximity of the quencher to the donormoiety on the probe. However, an understanding of the molecularmechanisms of the quenching phenomenon is not required to make or usethe invention.

If the solution containing the hybridization complex is heated (as inthe melting curve Tm analysis), the probe eventually dissociates fromthe target polynucleotide, thereby reducing the affinity of the quencherfor the nucleic acid, resulting in reduced proximity of the solublequencher to the probe donor and an increase in fluorescence from thedonor is observed. Thus, the formation/dissociation of hybridizationcomplexes in a reaction can be monitored by the use of a system having asoluble quencher.

Following formation of the duplex under conditions where base-pairingcan occur, the temperature of the target hybridization complex reactionis raised and the emission from the donor is measured and monitored overa range of temperatures, thus forming a melting curve. A temperaturerange of, for example, about 20° C. to about 95° C. can be used.Alternatively, the probe, soluble quencher and target can start at anelevated temperature (e.g., about 95° C.), and the donor emission ismonitored while the temperate of the reaction is lowered (e.g., to about20° C.), thus generating an annealing curve.

The Examples illustrating Tm determinations provided herein use asingle-labeled oligonucleotide probe, where the probe is labeled withFAM (6-carboxy-fluorescein), which serves as the light-emitting donormoiety in the donor/quencher pair with the soluble quencher. It will beapparent to one of skill that it is not intended that the presentinvention be limited to the use of FAM as the donor moiety. Indeed, theart it replete with descriptions of other label moieties, all of whichfind use with the invention as light emitting donor moieties. It isintended that these additional light emitting moieties also fall withinthe scope of the invention.

In either case of an annealing curve or a melting curve, the measuredemission from the donor is correlated with a particular duplexassociation/dissociation value, and a Tm is derived where the Tm is thattemperature at which one half of a population of hybridization complexesbecomes dissociated into single strands.

The invention provides numerous examples of Tm determinations usingsoluble quencher systems of the invention. For example, see Examples19-22. Many of the Examples provided herein utilize the soluble quencherreagents in viral genotyping methods, for example, HCV genotyping. Inthese methods, various viral genomic sequences are used as hybridizationtargets for a probe that is labeled with a light emitting moiety (e.g.,a donor such as FAM). Adaptation of these methods find particular use inviral detection and genotyping in clinical samples, for example, samplesfrom patients. However, it is not intended that the Tm determinationmethods of the invention be limited to viral genotyping applications.That is to say, it is not intended that the hybridization targets beviral material or nucleic acids derived from viral material. Indeed, awide variety of applications in addition to viral genotyping areimmediately apparent to one of skill in the art. For example, Tmdeterminations employing soluble quencher reagents can have use incoupled amplification, detection and analysis in a closed tube formatand using single labeled probes. These methods find uses in a widevariety of applications for example but not limited to such applicationsas SNP and mutation detection, haplotyping, microsatellite detection,characterization of pathogen genotypes, and characterization of drugresistant strains

The nature of the hybridization target is not particularly limited. Insome aspects, the hybridization target can be an amplicon, for example,an amplicon produced by a polymerase chain reaction. In some aspects,the PCR amplification can use an asymmetric amplification. In the casewhere the target nucleic acid of interest is an RNA molecule, the PCRamplification can employ reverse transcription PCR(RT-PCR).

Additional detailed description of general Tm methodologies and Tmdeterminations that utilize soluble light emission modifiers (i.e.,soluble quenchers) is found in cofiled U.S. patent application Ser. No.11/474,125, filed on Jun. 23, 2006, entitled “PROBES AND METHODS FORHEPATITIS C VIRUS TYPING USING SINGLE PROBE ANALYSIS,” by Gupta andWill; and also in cofiled U.S. patent application Ser. No. 11/474,092,filed on Jun. 23, 2006, entitled “PROBES AND METHODS FOR HEPATITIS CVIRUS TYPING USING MULTIDIMENSIONAL PROBE ANALYSIS,” by Gupta and Will.The entire content of these two cofiled applications are herebyincorporated by reference in their entirety for all purposes.

Kits for Tm Determination

The invention provides articles of manufacture, for example, kits tofacilitate the methods of the present invention, e.g., methods forconducting Tm determinations. These kits provide the materials necessaryfor making a Tm determination using the methods described herein. Thesekits find use for the clinician, who can use the Tm assessments, forexample, to make viral genotyping determinations. Materials and reagentsto carry out these methods can be provided in the kits to facilitateexecution of the methods.

In some embodiments, the Tm determination kits are diagnostic kits,where the information obtained from performing the methods enabled bythe kits is used, e.g., to identify the genotype of a virus in a sampletaken from a patient.

In certain embodiments, the invention provides kits suitable forconducting target amplification in addition to target Tm determination,for example, by incorporating PCR or RT-PCR reagents.

In some embodiments, the present invention provides kits for determiningthe melting temperature (Tm) of a particular hybridization complex,where the Tm determination uses a soluble light emission modifier (e.g.,a soluble quencher) system to monitor the duplex melting curve orannealing curve. These kits include, but are not limited to, (i) atleast one probe labeled with a suitable light emitting moiety (e.g., adonor), (ii) a soluble light emission modifier such as a thiazine dye ora diazine dye, where the dye is capable of quenching the light emittingmoiety, and (iii) one or more containers that hold the probe, thesoluble quencher, or both the probe and the soluble quencher.

Kits can also optionally include reagents for sample collection (e.g.,the collection of a blood sample), reagents for the collection andpurification of RNA from blood, a reverse transcriptase, primerssuitable for reverse transcription and first strand and second strandcDNA synthesis (i.e., reverse transcriptase initiation), e.g., toproduce a viral amplicon, a thermostable DNA-dependent DNA polymeraseand free deoxyribonucleotide triphosphates. In some embodiments, theenzyme comprising reverse transcriptase activity and thermostableDNA-dependent DNA polymerase activity are the same enzyme, e.g., Thermussp. ZO5 polymerase or Thermus thermophilus polymerase. The kits of theinvention can also optionally include standardization samples (e.g.,standardization nucleic acid templates at known concentrations to assessthe sensitivity of the Tm method); positive control samples (forexample, defined sequence nucleic acid templates with known, previouslydetermined Tm values), negative control samples (e.g., buffers orreaction mixtures that do not contain any nucleic acid target), bufferssuitable for enzymatic reactions, sample collection tubes andamplification reaction tubes.

Tm Determination Systems

In some embodiments, the invention provides integrated systems formaking Tm determinations. The systems can include instrumentation andmeans for interpreting and analyzing collected data, especially wherethe means for deriving the Tm comprise algorithms and/or electronicallystored information (e.g., collected fluorescence data, predetermined Tmcorrelations, etc). Each part of an integrated system is functionallyinterconnected, and in some cases, physically connected. In someembodiments, the integrated system is automated, where there is norequirement for any manipulation of the sample or instrumentation by anoperator following initiation of the Tm analysis.

A system of the invention can include instrumentation. For example, theinvention can include a detector such as a fluorescence detector (e.g.,a fluorescence spectrophotometer). A detector or detectors can be usedin conjunction with the invention, e.g., to monitor/measure the emissionfrom the light emitting moiety on the Tm probe. A detector can be in theform of a multiwell plate reader to facilitate the high-throughputcapacity of the Tm assay.

In some embodiments, the integrated system includes a thermal cyclingdevice, or thermocycler, for the purpose of controlling the temperatureof the Tm melting analysis. In some embodiments, the thermal cyclingdevice and the detector are an integrated instrument, where the thermalcycling and emission detection (e.g., fluorescence detection) are donein the same device.

A detector, e.g., a fluorescence spectrophotometer, can be connected toa computer for controlling the spectrophotometer operational parameters(e.g., wavelength of the excitation and/or wavelength of the detectedemission) and/or for storage of data collected from the detector (e.g.,fluorescence measurements during a melting curve analysis). The computermay also be operably connected to the thermal cycling device to controlthe temperature, timing, and/or rate of temperature change in thesystem. The integrated computer can also contain the “correlationmodule” where the data collected from the detector is analyzed and wherethe Tm of the target hybridization complex is determined(electronically). In some embodiments, the correlation module comprisesa computer program that calculates the Tm based on the fluorescencereadings from the detector, and in some cases, optionally derives viralgenotype information of an unknown sample based on the Tm result. Insome embodiments, the correlation module compares the T_(m) of theunknown sample with a database (or table) of Tm values for known viraltypes to make a correlation between the Tm of the unknown sample and theviral genotype of the unknown sample.

In some aspects, a system of the invention for the determination of a Tmof a hybridization complex comprises a reaction mixture (e.g., which mayor may not include a sample) that includes (i) a nucleic acid probecomprising a light emitting moiety that emits a signal; (ii) ahybridization target nucleic acid that is complementary or partiallycomplementary to the nucleic acid probe; and (iii) a thiazine dye or adiazine dye. The system also includes a thermal control device forregulating the temperature of the melting reaction over a range oftemperatures, where the range of temperatures includes a temperaturewhere essentially all probe molecules anneal with the hybridizationtarget at a given set of hybridization conditions, a temperature where50% of the target hybridization complexes are dissociated, and atemperature where essentially no probe molecules anneal with thehybridization target and essentially no hybridization complexes arepresent at the hybridization conditions. The system can further includea detector for measuring the signal from the melting reaction over therange of temperatures; and also a correlation module that is operablycoupled to the detector and receives signal measurements over the rangeof temperatures, where the correlation module correlates the signalintensity with the presence of a hybridization complex comprising theprobe and the hybridization target in admixture with the thiazine dye ordiazine dye as a function of temperature, thereby determining the T_(m)of the target hybridization complex. In some aspects, the light emittingmoiety on the probe is a FRET donor moiety.

Use of Thiazine Dyes for Duplex Stabilization

A variety of nucleic acid techniques suffer from sequence mismatches inthe amplification and/or detection of nucleic acids. The presentinvention provides solutions to this problem, where the inventionprovides methods for stabilizing nucleic acid duplexes. These methodsfor nucleic acid duplex stabilization are effective at stabilizingnucleic acid duplexes that contain single mismatch positions as well asduplexes with multiple mismatch positions. Indeed, the methods describedherein can even further stabilize perfectly matched nucleic acidduplexes. The further stabilization of perfectly matched duplexes willallow the preservation of intact nucleic acid duplexes under conditionswhere the duplex would otherwise dissociate.

As provided in the present disclosure, a number of compounds areidentified herein that can bind and significantly stabilize mismatchesin nucleic acid duplexes. These are members of the thiazine dye family,for example but not limited to, thionin (also known as thionine),methylene blue, new methylene blue, 1,9-dimethly methylene blue,methylene grene, azure A, azure B, azure C, and toluidine blue.

Any thiazine dye provided herein can be used in the duplex stabilizationmethods of the invention. Furthermore, in addition to the particularthiazine structures taught herein, related structural variants of thesemolecules that retain the essential stabilization property can also beused with the methods of the invention. Such related molecules areencompassed within the scope of the invention.

By using the thiazine dyes and related compounds as additives tohybridization reactions, nucleic acid amplification and detection can bevastly improved in those situations where polymorphism exists under theprobes and/or primers. These are in fact the most demanding applicationswhere the stabilization additive must not adversely affect the enzymaticactivity (e.g., PCR or RT-PCR amplification). Any applications where thestabilization of mismatches is required will benefit from theseprotocols. Furthermore, applications where there is improvedstabilization of a perfectly matched duplex can also benefit from theseprotocols. Any type of nucleic acid hybridization that yields a nucleicacid hybridization complex can benefit from the methods of theinvention. This includes, but is not limited to, for example, thehybridization of one or a plurality of enzymatically-extendable PCRprimers to a target sequence; the hybridization of any nucleic acidmolecules where the site of hybridization serves an initiation pointthat is effective to prime a nucleic acid extension reaction; thehybridization of a 5′-nuclease probe to a target; the hybridization ofany type of labeled (or unlabeled) probe to a target such as used insouthern blotting or northern blotting; and the use of nucleic acidprobes in any type of screening, such as in genomic library or cDNAlibrary screening. In some embodiments, the target nucleic acid moleculein the hybridization is an amplicon.

In some aspects, by using the thiazine dyes and related compounds asadditives to hybridization reactions, nucleic acid amplification anddetection can be vastly improved in those situations where polymorphismexists under the probes and/or primers, for example, in viral genotypinganalysis.

The invention provides numerous examples of duplex stabilization usingthiazine dyes. For example, see Examples 20-25. Many of the Examplesprovided herein utilize HCV or HIV viral model systems to illustrate theadvantageous properties of the methods of the invention with regard toduplex stabilization. However, it is not intended that the duplexstabilization methods of the invention be limited to viral hybridizationapplications. That is to say, it is not intended that the stabilizedduplexes comprise viral material or nucleic acids derived from viralmaterial. Indeed, a wide variety of other applications in addition toviral genetic analysis are immediately apparent to one of skill in theart.

Essentially, the methods for stabilizing the nucleic acid duplex consistof exposing the nucleic acid duplex to the stabilizing thiazine dye. Thethiazine dye can be admixed with the duplex at any point, for example,prior to formation of the duplex, or after formation of the duplex. Thethiazine can be present or absent during the annealing of the two ormore single strands of nucleic acid that form the duplex. In the casewhere the thiazine dye is absent during the formation of the duplex, thedye can be added after the duplex is formed.

The improved stability of the duplex using the stabilization methods ofthe invention can be observed by using any suitable assay to determineduplex stability. For example, the Tm of a nucleic acid duplex in theabsence of a thiazine dye can be compared to the Tm of the same nucleicacid duplex in the presence of the thiazine dye under the samehybridization conditions. Alternatively, a C_(T) growth curve (e.g., aC_(T) growth curve that uses a 5′-nuclease assay probe) can be conductedunder similar conditions, where the C_(T) value in the absence of thethiazine dye is compared to the C_(T) value in the presence of thethiazine dye. Note that when a C_(T) value is used to illustrate duplexstability, that C_(T) value can be a reflection of the duplex stabilityof either or both amplification primers, and furthermore, also reflectsthe stability of any probe-containing duplex that is used to monitor theamplicon accumulation (e.g., a 5′-nuclease probe in a 5′-nucleaseassay). It is significant to point out that when C_(T) determinationsare made to assess duplex stability, a 5′-nuclease assay need not beused to monitor amplicon accumulation. As illustrated in Example 25, aprobeless monitoring system can be used, such as by monitoring ampliconaccumulation using a double-stranded nucleic acid indicator such asSYBR® Green.

These methods for comparing duplex stability in dye-absent versusdye-present systems also apply to comparing the stability of duplexesthat are stabilized by any two different concentrations of a thiazinedye, for example, a high concentration and a low concentration.

The present invention provides methods for stabilizing nucleic acidduplexes, where the duplexes can be perfectly matched duplexes, orcontain any number of mismatch positions. For example, these methods fornucleic acid duplex stabilization are effective at stabilizing nucleicacid duplexes that contain one or more mismatch positions, two or moremismatch positions, or three or more mismatch positions.

In the methods for nucleic acid duplex stabilization, the concentrationof the thiazine dye that is used in the methods is not particularlylimited. In some aspects, a concentration of at least 10 μg/mL is used.In other aspects, any concentration within a range of concentrations isused, for example, a concentration of between about 10 μg/mL and about50 μg/mL. Alternatively, a concentration range of about 20 μg/mL andabout 40 μg/mL is used. In some aspects, a thiazine dye concentration ofabout 40 μg/mL is used.

Typically, in the methods for stabilizing nucleic acid duplexes, thestabilized hybridization complex is an intermolecular hybridizationcomplex, where the antiparallel hybridizing strands are two separatenucleic acid molecules. However, in some adaptations of the methods forstabilizing nucleic acid duplexes, the stabilized hybridization complexis an intramolecular hybridization complex, where the antiparallelhybridizing strands are actually on a single nucleic acid molecule, suchas in the case of a molecular beacon type configuration.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and are not intended to limit the scopeof the claimed invention. It is also understood that variousmodifications or changes in light the examples and embodiments describedherein will be suggested to persons skilled in the art and are to beincluded within the spirit and purview of this application and scope ofthe appended claims.

These examples show performance data obtained for certain 5′-nucleaseassays in which light emission modifiers of the present invention wereutilized.

Example 1 General Protocols for Fluorescence Quenching Assays

The tables included in this section describe the respective reactioncomponents, conditions, and procedures that were utilized in theanalyses referred to in the examples provided below, unless specifiedotherwise. In general, the fluorescence of labeled oligonucleotides wasmeasured in solutions both with and without a soluble quencher. Incertain analyses, for example, a series of probes labeled with FAM atthe 5′ end were measured in 400 μL solutions containing a PCR reactionbuffer (described below) and various concentrations of solublequenchers. For detecting the fluorescence of the FAM label in thoseanalyses, the wavelength of the excitation light was chosen to be 485nanometers and the fluorescence was measured at a wavelength of 520nanometers.

General Methods used for PCR Evaluations

TABLE III Reaction mixture for quenching analyses using a singlestranded fluorescent oligonucleotide: Component Concentration poly rAcarrier 9 μg/mL Glycerol 6.2% DMSO 7.5% Tricine, pH 8.3 50 mM KOAc 100mM dATP 300 μM each dCTP dGTP dUTP 550 μM SK145BU 0.4 μM (mockamplification primer) (40 pmol/rx) GAG152BU 0.4 μM (mock amplificationprimer) (40 pmol/rx) GAG108AF 0.1 μM (fluorescent oligonucleotide) UNG(Uracil N-Glycosylase) 10 U/reaction (rx) ZO5 DNA polymerase 40U/reaction EDTA 5 mM Mn(OAc)₂ 3 mM Light emission modifier 0-50 μg/mL

TABLE IV Reaction mixture for quenching analyses using a double strandedfluorescent oligonucleotide: Component Concentration poly rA carrier 9μg/mL Glycerol 6.2% DMSO 7.5% Tricine, pH 8.3 50 mM KOAc 100 mM dATP 300μM each dCTP dGTP dUTP 550 μM SK145BU 0.4 μM (mock amplification primer)(40 pmol/rx) GAG152BU 0.4 μM (mock amplification primer) (40 pmol/rx)GAG108AF 0.1 μM (fluorescent oligonucleotide) GAG100C 0.1 μM (complementto GAG108AF) UNG (Uracil N-Glycosylase) 10 U/reaction ZO5 DNA polymerase40 U/reaction EDTA 5 mM Mn(OAc)₂ 3 mM Light emission modifier 0-50 μg/mL

TABLE V Reaction mixture for quenching analyses using a fluorescentdinucleotide: Component Concentration poly rA carrier 9 μg/mL Glycerol6.2% DMSO 7.5% Tricine, pH 8.3 50 mM KOAc 100 mM dATP 300 μM each dCTPdGTP dUTP 550 μM SK145BU 0.4 μM (mock amplification primer) (40 pmol/rx)GAG152BU 0.4 μM (mock amplification primer) (40 pmol/rx) FAM-TT 0.1 μM(fluorescent dinucleotide) UNG (Uracil N-Glycosylase) 10 U/reaction ZO5DNA polymerase 40 U/reaction EDTA 5 mM Mn(OAc)₂ 3 mM Light emissionmodifier 0-50 μg/mL

Sequence Information

TABLE VI HCV Sequences SEQ ID Amplification primer Sequence NOST280ATBUA1 GCAGAAAGCGTCTAGCCATGGCGTTX 1 where X = N6-t-butylbenzyl-dAST778AATBA1 GCAAGCACCCTATCAGGCAGTACCACAX 2 where X = N6-t-butylbenzyl-dAST650AAFBHQ2 ECGGTGTACTCACCGJTTCCGCAGACCACTATGP 3 Quenched Where E= FAM; J = BHQ-2; P = Terminal 5′-Nuclease Probe PhosphateST650ACY5F14IN ECGGTGTACTCACCGJGTTCCGCAGACCACTATGP 4 Quenched where E= CY5; J = cx-FAM; P = Terminal 5′-Nuclease Probe Phosphate ST650A_5′-FLECGGTGTACTCACCGTTCCGCAGACCACTATGP 5 Single-labeled Probe where E = FAM;P = Terminal Phosphate

TABLE VII HIV Sequences SEQ ID Amplification primer Sequence NO SK145BUAGTGGGGGGACATCAAGCAGCCATGCAAX 6 where X = N6-t-butylbenzyl-dA GAG152BUGGTACTAGTAGTTCCTGCTATGTCACTTCX 7 where X = N6-t-butylbenzyl-dA GAG100CTAAAAGATACCATCAATGAGGAAGCTGCAGAP 8 (complement-GAG108) where P= Terminal Phosphate GAG108_5′-FAM ETCTGCAGCTTCCTCATTGATGGTATCTTTTAP 9Single-labeled probe where E = FAM; P = Terminal Phosphate

TABLE VIII Quantitation standard (QS) SEQ ID Amplification primerSequence NO ST280ATBUA1 GCAGAAAGCGTCTAGCCATGGCGTTX 10 where X= N6-t-butylbenzyl-dA ST778AATBA1 GCAAGCACCCTATCAGGCAGTACCACAX 11 whereX = N6-t-butylbenzyl-dA ST2535_5-HEX Single-ETGGACTCAGTCCTCTGGTCATCTCACCTTCTP 12 labeled Probe where E = HEX; P= Terminal Phosphate

TABLE IX Reaction mixture for HCV PCR using a single-labeled fluorescentprobe Component Concentration poly rA carrier 9 μg/mL Glycerol 6.2% DMSO7.5% Tricine, pH 8.3 50 mM KOAc 100 mM dATP 300 μM each dCTP dGTP dUTP550 μM ST280ATBUA1 0.4 μM amplification primer (40 pmol/rx) ST778AATBA10.4 μM amplification primer (40 pmol/rx) ST650_5′-FAM 0.1 μM(single-labeled fluorescent probe) (10 pmol/rx) UNG (UracilN-Glycosylase) 10 U/reaction ZO5 DNA polymerase 40 U/reaction Mn(OAc)₂ 3mM Light emission modifier 0-50 μg/mL HCV TARGET DNA 2-10⁶ copies perreaction

TABLE X Reaction mixture for HCV PCR using a quenched 5′-nuclease probeComponent Concentration poly rA carrier 9 μg/mL Glycerol 6.2% DMSO 7.5%Tricine, pH 8.3 50 mM KOAc 100 mM dATP 300 μM each dCTP dGTP dUTP 550 μMST280ATBUA1 0.4 μM (amplification primer) (40 pmol/rx) ST778AATBA1 0.4μM (amplification primer) (40 pmol/rx) ST650AAFBHQ2 0.1-0.2 μM orST650ACY5F14IN (10-20 pmol/rx) (Quenched 5′-nuclease probe) UNG (UracilN-Glycosylase) 10 U/reaction ZO5 DNA polymerase 40 U/reaction Mn(OAc)₂ 3mM Light emission modifier 0-50 μg/mL HCV TARGET DNA 2-10⁶ copies perreaction

TABLE XI Reaction Mixture for HIV PCR Using a Single-Labeled FluorescentProbe Component Concentration poly rA carrier 9 μg/mL Glycerol 6.2% DMSO7.5% Tricine, pH 8.3 50 mM KOAc 100 mM dATP 300 μM each dCTP dGTP dUTP550 μM SK145BU 0.4 μM (amplification primer) (40 pmol/rx) GAG152BU 0.4μM (amplification primer) (40 pmol/rx) GAG108AF 0.1 μM (single-labeledfluorescent probe) UNG (Uracil N-Glycosylase) 10 U/reaction ZO5 DNApolymerase 40 U/reaction Mn(OAc)₂ 3 mM Light emission modifier 0-50μg/mL HIV target DNA 2-10⁶ copies per reaction

TABLE XII Reaction mixture for HCV RT-PCR using a single-labeledfluorescent probe Component Concentration poly rA carrier 9 μg/mLGlycerol 6.2% DMSO 7.5% Tricine, pH 8.3 50 mM KOAc 100 mM dATP 300 μMeach dCTP dGTP dUTP 550 μM ST280ATBUA1 0.4 μM amplification primer (40pmol/rx) ST778AATBA1 0.4 μM amplification primer (40 pmol/rx)ST650AAFBHQ2 0.1-0.2 μM Or ST650ACY5F14IN (10-20 pmol/rx) Quenched5′-nuclease Probe UNG (Uracil N-Glycosylase) 10 U/reaction ZO5 DNApolymerase 40 U/reaction Mn(OAc)₂ 3 mM Light emission modifier 0-50μg/mL HCV TARGET RNA 2-10⁶ copies per reaction

TABLE XIII Reaction mixture for HIV RT-PCR using a single-labeledfluorescent probe Component Concentration poly rA carrier 9 μg/mLGlycerol 6.2% DMSO 7.5% Tricine, pH 8.3 50 mM KOAc 100 mM dATP 300 μMeach dCTP dGTP dUTP 550 μM SK145BU 0.4 μM (amplification primer) (40pmol/rx) GAG152BU 0.4 μM (amplification primer) (40 pmol/rx) GAG108AF0.1 μM (single-labeled fluorescent probe) UNG (Uracil N-Glycosylase) 10U/reaction ZO5 DNA polymerase 40 U/reaction Mn(OAc)₂ 3 mM Light emissionmodifier 0-50 μg/mL HIV target RNA 2-10⁶ copies per reaction

Thermocycling Conditions

TABLE XIV HCV and QS PCR Thermocycling Stage 1 50° C./5 m Stage 2 95°C./2 m Stage 3 95° C./15 s ↓  2 cycles 58° C./50 s Stage 4 95° C./15 s ↓60 cycles 50-58° C./50 s Stage 5 4° C./inf.

TABLE XV HCV RT-PCR Thermocycling Stage 1 50° C./5 m Stage 2 59° C./30 mStage 3 95° C./2 m Stage 4 95° C./15 s ↓  2 cycles 50-58° C./50 s Stage5 95° C./15 s ↓ 60 cycles 58° C./50 s Stage 6 4° C./inf.

TABLE XVI HIV PCR Thermocycling Stage 1 50° C./5 m Stage 2 95° C./2 mStage 3 95° C./15 s ↓  2 cycles 58° C./50 s Stage 4 91° C./15 s ↓ 60cycles 50-58° C./50 s Stage 5 4° C./inf.

TABLE XVII HIV RT-PCR Thermocycling Stage 1 50° C./5 m Stage 2 59° C./30m Stage 3 95° C./2 m Stage 4 95° C./15 s ↓  2 cycles 50-58° C./50 sStage 5 91° C./15 s ↓ 60 cycles 58° C./50 s Stage 6 4° C./inf.

Example 2 Single-Labeled Probes Fluorescence Quenching

This Example and Examples that follow illustrate various performancecharacteristics of assays that included the use of light emissionmodifiers described herein and assorted single-labeled probes. Thisexample illustrates the quenching of fluorescence with various lightemission modifiers of the invention.

FIG. 4 is a graph (ordinate represents percent fluorescence, abscissarepresents concentration (μg/mL)) that shows the quenching offluorescence from different single-labeled nucleic acids with variousconcentrations of new methylene blue. As shown in the legend thataccompanies the graph, the plots are for a single-labeled,single-stranded oligonucleotide having a sequence of 31 nucleotides(i.e., a 31-mer (GAG108AF; ETCTGCAGCTTCCTCATTGATGGTATCTTTTAP, whereE=FAM and P=phosphate (SEQ ID NO: 13))), a single-labeled,double-stranded 31-mer, and a single-labeled dinucleotide or dimer atthe indicated new methylene blue concentrations. Each of these nucleicacids included a 5′-end FAM label.

FIG. 5 is a graph (ordinate represents percent fluorescence, abscissarepresents light emission modifier concentration (μg/mL)) that shows thequenching of fluorescence using various light emission modifiersperformed in separate analyses. Single-stranded 31-mers comprising5′-end FAM labels were used in these analyses (i.e., GAG108AF, above).As shown in the legend accompanying the plot, the different lightemission modifiers utilized were methylene blue (Me Blue), new methyleneblue (N Me Blue), azure B, thionin, dimethyl methylene blue (DM MeBlue), and toluidine blue (T Blue).

FIG. 6 is a graph (ordinate represents percent fluorescence, abscissarepresents light emission modifier concentration (μg/mL)) that shows thequenching of fluorescence from single-labeled, double-strandedoligonucleotides with various light emission modifiers. That is, the5′-ends of one strand of the double-stranded 31-mers used in theseseparate analyses were labeled with FAM. As shown in the legendaccompanying the plot, the different light emission modifiers utilizedwere methylene blue (Me Blue), new methylene blue (N Me Blue), azure B,thionin, dimethyl methylene blue (DM Me Blue), and toluidine blue (TBlue).

FIG. 7 is a graph (ordinate represents percent fluorescence, abscissarepresents light emission modifier concentration (μg/mL)) that shows thequenching of fluorescence from labeled dinucleotides (thymidine dimers(TT)) with various light emission modifiers. The dinucleotide used inthese analyses was labeled at 5′-ends with FAM (i.e.,6-carboxy-fluorescein). As shown in the legend accompanying the plot,the different light emission modifiers utilized were methylene blue (MeBlue), new methylene blue (N Me Blue), azure B, thionin, dimethylmethylene blue (DM Me Blue), and toluidine blue (T Blue).

FIG. 8 is a graph (ordinate represents percent fluorescence, abscissarepresents light emission modifier concentration (μg/mL)) that shows thequenching of fluorescence from labeled, single-stranded oligonucleotideswith methylene blue under various conditions. Single-stranded 31-merscomprising 5′-end FAM labels were used in these analyses. As shown inthe legend accompanying the plot, the reaction mixtures represented byone trace included poly rA, whereas the reaction mixtures represented bythe other trace lacked poly rA. Poly rA is adenosine homopolymer that isgenerally used as a component of a sample diluent buffer. It serves as acarrier nucleic acid, and improves the sensitivity of the assays byminimizing losses of target nucleic acids after sample preparation. PolyrA is typically used at a relatively high concentration. The analysisillustrated in FIG. 8 evaluated whether poly rA interferes with theeffectiveness of a soluble quencher, e.g., by binding to it and makingit less available.

Example 3 Polymerase Chain Reactions Using Single Label Probes and AzureDyes

This example illustrates the embodiment describing real time detectionwith a single-labeled probe and a light emission modifier of theinvention.

FIG. 9 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labeled5′-nuclease probes and various concentrations of azure B in HCVdetection assays. The single-labeled ST650 probes (corresponding to SEQID NO: 5) used in the reaction mixtures represented by these traces werelabeled at 5′-ends with FAM. The reaction mixtures also included polyrA. The labels accompanying the traces show the concentration of azure Bused in each of these reaction mixtures. The relative fluorescence as afunction of light emission modifier concentration for these reactions isplotted in FIG. 11. This plot shows, e.g., that relative fluorescenceincreases with increasing light emission modifier concentration.

FIG. 10 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labeled5′-nuclease probes and various concentrations of azure B in HIVdetection assays. The single-labeled ST650 probes (corresponding to SEQID NO: 5) used in the reaction mixtures represented by these traces werelabeled at 5′-ends with FAM. The reaction mixtures also included polyrA. The labels accompanying the traces show the concentration of azure Bused in each of these reaction mixtures. As shown, when the HCV probewas used in an HIV amplification system, the probe was not cleaved andno growth curves were observed. This analysis showed the observed growthcurve to be specific to probe hydrolysis and not due to the partitioningof the soluble quencher into the amplicon.

Example 4 Polymerase Chain Reactions Using Single Label Probes and NewMethylene Blue

FIG. 12 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labelednuclease probes and new methylene blue in HCV detection assays. Thesingle-labeled ST650 probes (corresponding to SEQ ID NO: 5) used inthese reaction mixtures were labeled at 5′-ends with FAM. In addition,the reaction mixtures represented by these traces included poly rA andthe denaturing temperature (T_(den)) used in these reactions was 95° C.20,000 copies of HCV cDNA were present in each reaction mixture. Theannealing temperature used in these reactions was 58° C. The newmethylene blue concentrations used in these reaction mixtures areindicated by the labels that accompany the plot. The amplification plotof FIG. 13 shows the relative fluorescence for this data.

Example 5 Polymerase Chain Reactions Using Double Label Probes and1,9-Demethyl Methylene Blue

FIG. 14 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included dual-labeled5′-nuclease probes and 1,9-dimethyl methylene blue in HCV detectionassays. The dual-labeled ST650 probes (corresponding to SEQ ID NO: 5)used in these reaction mixtures were labeled with FAM and a BHQ™. Inaddition, the reaction mixtures represented by these traces includedpoly rA and the T_(den) used in these reactions was 95° C. 20,000 copiesof HCV cDNA were present in each reaction mixture. The annealingtemperature used in these reactions was 58° C. The new methylene blueconcentrations used in these reaction mixtures are indicated in thelabels that accompany the plot. The amplification plot of FIG. 15 showsthe relative fluorescence for this data.

Example 6 Polymerase Chain Reactions Using Single Label Probes and AzureA and C

FIG. 16 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labeled5′-nuclease probes and azure A in HCV detection assays. Thesingle-labeled ST650 probes (corresponding to SEQ ID NO: 5) used inthese reaction mixtures were labeled at 5′-ends with FAM. Further, thereaction mixtures represented by these traces included poly rA and theT_(den) used in these reactions was 95° C. 20,000 copies of HCV cDNAwere present in each reaction mixture. The azure A concentrations usedin these reaction mixtures are indicated in the labels that accompanythe plot.

FIG. 17 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labelednuclease probes and azure C in HCV detection assays. The single-labeledST650 probes (corresponding to SEQ ID NO: 5) used in these reactionmixtures were labeled at 5′-ends with FAM. In addition, the reactionmixtures represented by these traces included poly rA and the T_(den)used in these reactions was 95° C. 20,000 copies of HCV cDNA werepresent in each reaction mixture. The azure C concentrations used inthese reaction mixtures are indicated in the labels that accompany theplot.

Example 7 Polymerase Chain Reactions Using Single Label Probes andThionin

FIG. 18 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labelednuclease probes and thionin in HCV detection assays. The single-labeledST650 probes (corresponding to SEQ ID NO: 5) used in these reactionmixtures were labeled at 5′-ends with FAM. Further, the reactionmixtures represented by these traces included poly rA and the T_(den)used in these reactions was 95° C. 20,000 copies of HCV cDNA werepresent in each reaction mixture. The thionin concentrations used inthese reaction mixtures are indicated in the labels that accompany theplot.

Example 8 Polymerase Chain Reactions Using Single Label Probes andMethylene Green

FIG. 19 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labeled5′-nuclease probes and methylene green in HCV detection assays. Thesingle-labeled ST650 probes (corresponding to SEQ ID NO: 5) used inthese reaction mixtures were labeled at 5′-ends with FAM. In addition,the reaction mixtures represented by these traces included poly rA andthe T_(der) used in these reactions was 95° C. 20,000 copies of HCV cDNAwere present in each reaction mixture. The methylene greenconcentrations used in these reaction mixtures are indicated in thelabels that accompany the plot.

Example 9 Polymerase Chain Reactions Using Single Label Probes andVarious Light Emission Modifiers

FIG. 20 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labeled5′-nuclease probes and different azure dyes in HCV detection assays. Thesingle-labeled ST650 probes (corresponding to SEQ ID NO: 5) used inthese reaction mixtures were labeled at 5′-ends with FAM. The reactionmixtures represented by these traces included poly rA and 200,000 copiesof a target nucleic acid from HCV. The T_(den) used in these reactionswas 95° C. As shown in the labels that accompany the plot, the dyes usedin these analyses were azure A, azure B, and azure C, which were eachpresent in the respective reaction mixtures at a concentration of 40μg/mL.

FIG. 21 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labeled5′nuclease probes and various light emission modifiers in HCV detectionassays. The single-labeled ST650 probes (corresponding to SEQ ID NO: 5)used in these reaction mixtures were labeled at 5′-ends with FAM. Thereaction mixtures represented by these traces included poly rA and200,000 copies of a target nucleic acid from HCV. The T_(den) used inthese reactions was 95° C. As shown in the labels that accompany theplot, the light emission modifiers used in these analyses were azure A,azure B, azure C, methylene blue, toluidine blue, thionin, and methylenegreen, which were each present in the respective reaction mixtures at aconcentration of 40 μg/mL.

Example 10 Polymerase Chain Reactions Using Various Concentrations ofMethylene Blue

FIG. 22 (panels A and B) is a photograph of a polyacrylamide gelanalysis of PCR reactions with target HCV DNA, various probes, andvarious amounts of methylene blue. The numbers shown above the lanes inthe gel indicate the concentrations (μg/mL) of methylene blue that wereincluded in the particular reaction mixtures. Lanes denoted with 0(−)are those in which negative controls were run. The reaction mixturesincluded poly rA and 20,000 copies of the target HCV DNA. In addition,the T_(den) used in these reactions was 95° C. As shown, the probesutilized were ST650_(—)5′-FAM & ST2325_(—)5′-HEX, dual ST650, and dualST2535, which are described above. Panels A and B represent duplicatereactions. This analysis showed that PCR amplification is relativelyunaffected by the presence of increasing amounts of the light emissionmodifier.

FIG. 23 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labeled5′-nuclease probes and methylene blue in QS-HCV detection assays. Thesingle-labeled ST2535 probes (corresponding to SEQ ID NO: 12) used inthese reaction mixtures were labeled at 5′-ends with HEX. The reactionmixtures represented by these traces included poly rA and the T_(den)used in these reactions was 95° C. 20,000 copies of QS HCV cDNA werepresent in each reaction mixture. The methylene blue concentration usedin these reaction mixtures was 40 μg/mL. The labels that accompany theplot indicate the concentration of methylene blue used in the reactionmixtures represented by each trace. This analysis demonstrated, e.g.,the ability of thiazine dyes to quench different fluorophores (e.g.,other than FAM).

FIG. 24 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included multiplesingle-labeled nuclease probes and methylene blue in QS-HCV detectionassays. The single-labeled probes used in these multiplexing analyseswere 5′-FAM and 5′-HEX labeled oligonucleotides. The reaction mixturesrepresented by these traces included poly rA and the T_(den) used inthese reactions was 95° C. 20,000 copies of QS HCV cDNA were present ineach reaction mixture. The methylene blue concentrations used in thereaction mixtures with these probe pairs is indicated in the labels thataccompany the plot. This analysis illustrates the ability of thiazinedyes to be used in multiplex detection with single-labeled probes.

Example 11 Polymerase Chain Reactions Using Azure B

FIG. 25 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from 5′-nuclease reactions performed in the absence of azure Bin HCV detection assays. The reaction mixtures represented by thesetraces lacked azure B and included 20,000 copies of a target nucleicacid from HCV. The denaturing temperature (T_(den)) used in thesereactions was 95° C. As shown in the accompanying trace labels, thereaction mixtures included either probes labeled at 5′-ends with FAM(i.e., 5′-FAM) or dual labeled probes (i.e., dual) and either includedor lacked poly rA. As shown, there was a slight release of fluorescencefrom the unquenched single-labeled probe. This may have been due to someG-quenching in the probe. However, this signal increase is generally tooinsignificant to be useful in a practical assay. In contrast, the duallabeled probe generated a good signal. Further, in the presence of thesoluble quencher, the single-labeled probe also generated a good signal(see, e.g., FIGS. 26 and 27, which are described below).

FIGS. 26 and 27 are amplification plots (ordinate represents absolutefluorescence (FIG. 26), or normalized fluorescence (FIG. 27), abscissarepresents the cycle number) that shows data obtained from various5′-nuclease reactions that included azure B in HCV detection assays. Thereaction mixtures represented by these traces included 30 μg/mL of azureB and 20,000 copies of a target nucleic acid from HCV. The T_(den) usedin these reactions was 95° C. As shown in the accompanying trace labels,the reaction mixtures included either probes labeled at 5′-ends with FAM(i.e., 5′-FAM) or dual labeled probes (i.e., dual) and either includedor lacked poly rA.

Example 12 Additional Amplification Reactions Using Methylene Blue

FIGS. 28 and 29 are amplification plots (ordinate represents relativefluorescence, abscissa represents the cycle number) that respectivelyshow data obtained from DNA and RNA template titrations that includedsingle-labeled nuclease probes and methylene blue in HCV detectionassays. The single-labeled ST650 probes (corresponding to SEQ ID NO: 5)used in these reaction mixtures were labeled at 5′-ends with FAM. Thereaction mixtures represented by these traces included poly rA and theT_(den) used in these reactions was 95° C. The methylene blueconcentration used in these reaction mixtures was 40 μg/mL. The labelsthat accompany the plots indicate the number of copies of the targetcDNA or RNA from HCV along with other reaction conditions for eachtrace. These analyses illustrate, e.g., that highly sensitive andquantitative PCR and RT-PCR detection can be achieved using thesemethods.

FIG. 30 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labeled5′-nuclease probes and methylene blue in HIV detection assays. Thesingle-labeled GAG108 probes (corresponding to SEQ ID NO: 9) used inthese reaction mixtures were labeled at 5′-ends with FAM. In addition,the reaction mixtures represented by these traces included poly rA andthe T_(den) used in these reactions was 95° C. The methylene blueconcentration used in the reaction mixtures was 40 μg/mL. The HCV cDNAcopy number that was present in each reaction mixture is indicated inthe labels that accompany the plot. This analysis illustrates, e.g.,that highly sensitive and quantitative PCR detection can be achievedusing this method.

FIG. 31 is a photograph of an agarose gel that shows the sensitivity ofdetection in 5′-nuclease assays in which target nucleic acids copynumbers were varied in the presence of methylene blue. The numbers shownabove the lanes in the gel indicate the target nucleic acid copy numberused for the particular run. Lanes denoted with (−) or no dye(−) arethose in which negative controls were run. The reaction mixturesincluded poly rA and the denaturing temperature (T_(den)) used in thesereactions was 95° C. The concentration of methylene blue in the reactionmixtures was 40 μg/mL. The target nucleic acid was from HCV and theprobe was ST650 (corresponding to SEQ ID NO: 5) in the reactions shownin panel A. The target nucleic acid was from HIV and the probe wasGAG108 (corresponding to SEQ ID NO: 9) in the reactions shown in panelB. These assays show, e.g., that there is no significant deleteriouseffect on PCR efficiency and detection sensitivity in the presence ofthe soluble quencher.

Example 13 Polymerase Chain Reactions Using Single-Labeled Probe and NewMethylene Blue

FIG. 32 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labeled5′-nuclease probes and new methylene blue in HCV detection assays. Thesingle-labeled ST650 probes (corresponding to SEQ ID NO: 5) used inthese reaction mixtures were labeled at 5′-ends with FAM. In addition,the reaction mixtures represented by these traces included poly rA andthe T_(den) used in these reactions was 95° C. 20,000 copies of HCV cDNAwere present in each reaction mixture. The annealing temperature used inthese reactions was 40° C. The new methylene blue concentrations used inthese reaction mixtures are indicated in the labels that accompany theplot.

FIG. 33 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included 5′-FAM labelednuclease probes and new methylene blue. This plot along with othersreferred to herein show the relative fluorescence signal modulation withtemperature. More specifically, fluorescence was detected at anannealing temperature of 40° C. in these reaction mixtures. Thesingle-labeled ST650 probes (corresponding to SEQ ID NO: 5) used inthese reaction mixtures were labeled at 5′-ends with FAM. Further, thereaction mixtures represented by these traces included poly rA and theT_(den) used in these reactions was 95° C. 20,000 copies of HCV cDNAwere present in each reaction mixture. The new methylene blueconcentrations used in these reaction mixtures is indicated in thelabels that accompany the plot.

FIG. 34 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from various 5′-nuclease reactions that included single-labeled5′-nuclease probes and new methylene blue in which different annealtemperatures where utilized. The single-labeled ST650 probes(corresponding to SEQ ID NO: 5) used in these reaction mixtures werelabeled at 5′-ends with FAM. In addition, the reaction mixturesrepresented by these traces included poly rA and the T_(den) used inthese reactions was 95° C. 20,000 copies of HCV cDNA were present ineach reaction mixture. The new methylene blue concentrations used in thereaction mixtures was 40 μg/mL. The annealing temperature associatedwith each reaction mixture is indicated in the labels that accompany theplot.

Example 14 Polymerase Chain Reactions Using Multiply-Labeled Probe andMethylene Blue

This example and other examples below illustrates the modification offluorescence in 5′-nuclease assays that included the use of multiplylabeled probes. This example shows the use of methylene blue in5′-nuclease reactions to modify the baseline emission of light from5′-nuclease probes.

FIG. 35 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from 5′-nuclease reactions performed in the presence of variousmethylene blue concentrations. The probes were each labeled with FAM anda BHQ™. The particular methylene blue concentrations are shown in thelabels that accompany the plot.

FIG. 36 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from 5′-nuclease reactions performed under the same conditionsused for the reactions described with respect to FIG. 35 aside fromdoubling the concentration of probes utilized in each reaction. Theparticular methylene blue concentrations are shown on the plot.

FIG. 37 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from 5′-nuclease reactions that simulated probe pooling. Theparticular methylene blue concentration and relative probe concentrationassociated with each trace are shown on the plot.

FIG. 38 is an amplification plot (ordinate represents relativefluorescence, abscissa represents the cycle number) that shows dataobtained from 5′-nuclease reactions performed using a FAM-BHQdual-labeled probe in the presence of various methylene blueconcentrations. The particular methylene blue concentration associatedwith each trace is shown in the labels that accompany the plot.

Example 15 Polymerase Chain Reactions Using Multiply-Labeled Probe and1,9-Dimethyl Methylene Blue

This example illustrates the use of 1,9-dimethyl methylene blue in5′-nuclease reactions to modify the baseline emission of light from5′-nuclease probes.

FIG. 39 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from 5′-nuclease reactions performed in the presence of various1,9-dimethyl methylene blue concentrations. The reaction mixturesincluded poly rA and the T_(den) used in these reactions was 95° C. Thereaction mixtures include 20,000 copies of a target nucleic acid fromHCV. The ST650 probe (corresponding to SEQ ID NO: 3) was labeled withFAM and BHQ™. The annealing temperature used in these reactions was 58°C. The particular 1,9-dimethyl methylene blue concentrations are shownin the labels that accompany the plot. The amplification plot of FIG. 40shows the relative fluorescence for this data.

Example 16 Polymerase Chain Reactions Using Multiply-Labeled Probe andNew Methylene Blue

This example illustrates the use of new methylene blue in 5′-nucleasereactions to modify the baseline emission of light from 5′-nucleaseprobes.

FIG. 41 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows dataobtained from 5′-nuclease reactions performed in the presence of variousnew methylene blue concentrations. The reaction mixtures included polyrA and the Td, used in these reactions was 95° C. The reaction mixturesinclude 20,000 copies of a target nucleic acid from HCV. The ST650 probe(corresponding to SEQ ID NO: 3) was labeled with FAM and a BHQ™. Theparticular new methylene blue concentrations are shown in the labelsthat accompany the plot. The annealing temperature used in thesereactions was 58° C. The amplification plot of FIG. 42 shows therelative fluorescence for this data.

Example 17 Quantitation Standard Amplification and Detection UsingHEX-Labeled Probe

This example illustrates the use of methylene blue to modify thebaseline emission of light from a HEX labeled 5′-nuclease probe in HCVquantitation standard (HCV-QS) amplification and detection assays. TheHCV QS DNA contained HCV primer binding sequences, and a uniqueQS-specific probe binding region. The reaction mixtures contained aprimer pair that is specific for HCV and HCV QS DNA, and detection ofthe amplified DNA was performed by measuring the emission intensity offluorescent reporter dyes released from the target specific QS probesduring amplification, which permitted independent identification of HCVand HCV QS.

More specifically, FIG. 43 is an amplification plot (ordinate representsraw fluorescence, abscissa represents the cycle number) that shows dataobtained from 5′-nuclease reactions performed in the presence of variousmethylene blue concentrations. The reaction mixtures included poly rAand the T_(den) used in these reactions was 95° C. The reaction mixturesincluded ST2535CY5H14 probes (corresponding to SEQ ID NO: 12) and 20,000copies of QS-DNA. The particular new methylene blue concentrations usedare shown in the labels that accompany the plot. The amplification plotof FIG. 44 shows the relative fluorescence for this data.

Example 18 Modification of Baseline Emission of Light in the Detectionof HCV Nucleic Acids

This example illustrates the use of various light emission modifiers tomodify the baseline emission of light from 5′-nuclease probes in assaysthat involved the detection of HCV nucleic acids.

FIG. 45 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows thedetection of HCV nucleic acids in 5′-nuclease reactions performed in thepresence of various Janus Green B concentrations. The reaction mixturesincluded ST650ACY5F14IN probes (corresponding to SEQ ID NO: 4) and20,000 copies of target HCV nucleic acids. The particular Janus Green Bconcentrations used are shown in the labels that accompany the plot.

FIG. 46 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows thedetection of HCV nucleic acids in 5′-nuclease reactions performed in thepresence of various toluidine blue concentrations. The reaction mixturesincluded ST650ACY5F14IN probes (corresponding to SEQ ID NO: 4) and20,000 copies of target HCV nucleic acids. The particular toluidine blueconcentrations used are shown in the labels that accompany the plot.

FIG. 47 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows thedetection of HCV nucleic acids in 5′-nuclease reactions performed in thepresence of various Victoria Pure Blue BO concentrations. The particularVictoria Pure Blue BO concentrations used are shown in the labels thataccompany the plot.

FIG. 48 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows thedetection of HCV nucleic acids in 5′-nuclease reactions performed in thepresence of various azure A concentrations. The particular azure Aconcentrations used are shown in the labels that accompany the plot.

FIG. 49 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows thedetection of HCV nucleic acids in 5′-nuclease reactions performed in thepresence of various methylene green concentrations. The reactionmixtures included poly rA and the T_(den) used in these reactions was95° C. The reaction mixtures included ST650ACY5F14IN probes(corresponding to SEQ ID NO: 4) and 20,000 copies of target HCV nucleicacids. The particular methylene green concentrations used are shown inthe labels that accompany the plot.

FIG. 50 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows thedetection of HCV nucleic acids in 5′-nuclease reactions performed in thepresence of various thionin concentrations. The reaction mixturesincluded poly rA and the T_(den) used in these reactions was 95° C. Thereaction mixtures included ST650ACY5F14IN probes (corresponding to SEQID NO: 4) and 20,000 copies of target HCV nucleic acids. The particularthionin concentrations used are shown in the labels that accompany theplot.

FIG. 51 is an amplification plot (ordinate represents absolutefluorescence, abscissa represents the cycle number) that shows thedetection of HCV nucleic acids in 5′-nuclease reactions performed in thepresence of various azure B concentrations. The reaction mixturesincluded poly rA and the T_(den) used in these reactions was 95° C. Thereaction mixtures included ST650ACY5F14IN probes (corresponding to SEQID NO: 4) and 20,000 copies of target HCV nucleic acids. The particularazure B concentrations used are shown in the labels that accompany theplot.

Example 19 Melting Curve Analysis (Tm Determination) Using aSingle-Labeled Probe in Conjunction with a Soluble Light EmissionModifier in an HCV Model System

The present example describes a melting curve analysis (i.e., Tmdetermination) using a single-labeled probe in conjunction with asoluble light emission modifier (i.e., soluble quencher). An HCV probeand HCV synthetic templates are used in the experimental system. Theeffectiveness of using the single-labeled probe with a soluble quencheris demonstrated.

An HCV typing probe was designed and synthesized (SEQ ID NO: 14), andcontains a single fluorescein (FAM) label, as shown in FIG. 52A. Thisprobe hybridizes to a domain within the heterogeneous 5′-UTR of the HCVgenome. The probe was alternatively hybridized with different syntheticsingle-stranded templates corresponding to various HCV genotypes aslisted in the table below.

HCV/ Genotype/ SEQ ID Subtype Synthetic Template NO: 1a/bAGGACCCGGTCGTCCTGGCAATTCCGGTGTA 15 2a/c AGGACCCAGTCTTCCCGGCAATTCCGGTGTA16 4 AGGACCCGGTCATCCCGGCGATTCCGGTGTA 17 2bAGGACCCGGTCTTTCCGGTAATTCCGGTGTA 18 5 AGGACCCGGTCATCCCGGCAATTCCGGTGTA 196 AGGACCCGGTCATCCTGGCAATTCCGGTGTA 20 3a AGGACCCGGTCACCCCAGCGATTCCGGTGTA21

The probe was annealed to each of the synthetic templates in separatereactions. For the melting analysis, the various hybridization mixtureswere heated to 95° C. for 2 min, followed by cooling to 20° C. to allowannealing and the formation of hybridization complexes. The reactionscontaining the hybridization complexes were then heated in approximately76 cycles where each cycle increases the temperature 1° C. for 30seconds. Fluorescence was measured for 50 milliseconds at the end ofeach 30 second cycle. The melting reactions were run in 96 wellmicrotiter plates, and fluorescence was monitored using an ABI PRISM®RTM 7700 Sequence Detection System (Applied Biosystems, Foster City,Calif.). Fluorescence was measured in this experiment (and allexperiments that used FAM-labelled probes) using an excitation filter at485 nm with a 20 nm bandwidth, and an emission filter at 520 nm with a10 nm bandwidth.

The formation/dissociation of hybridization complexes in the mix wasmonitored using a soluble quencher system. The FAM label covalentlyattached to the probe provided a suitable donor emission. The quenchingaction was provided by the soluble quencher methylene blue. Methyleneblue is a member of a family of soluble quenchers based on thiazine anddiazine dye structures. The methylene blue quencher has a bindingaffinity for double-stranded DNA, and exhibits a quenching effect whenin close proximity to the fluorescent label on the probe when the probeis in a duplex structure with the target. However, the soluble quencherhas reduced affinity for single-stranded DNA. Thus, when the solutioncontaining the hybridization complex comprising the probe is heated andeventually dissociates, the affinity of the quencher for the nucleicacid is reduced, resulting in an increase in fluorescence.

The fluorescence data can be shown graphically by plotting a rawfluorescence value as a function of temperature. In one controlexperiment, the methylene blue soluble quencher was omitted from themelting reaction. The results of separate experiments (a meltinganalysis using the probe and each HCV synthetic template) were overlaidon the same plot, and are shown in FIG. 52B. In these experiments, theresults of multiple separate experiments are overlaid on the same graph.A representative set of data is shown. As might be expected in theabsence of the soluble quencher, there was no significant change in FAMfluorescence indicating a transition from duplex to single strandedstate in each of the examples, due to the absence of a quenching moiety,despite the temperature cycling program which would result in annealingand melting of DNA duplexes.

Next, using the same reagents in a new analysis, methylene blue wasadded to the melting reactions at a concentration of 10 μg/mL. Theresults of that melting analysis are shown in FIG. 53. As can be seen,each probe/template complex gave a distinct dissociation profile uponheating, indicating varying Tm values for the different genotypes.

In the next experiment, methylene blue was added to the meltingreactions at a concentration of 20 μg/mL. The results of that meltinganalysis are shown in FIG. 54. As can be seen, each probe/templatecomplex again gave a distinct dissociation profile upon heating,indicating distinct Tm values corresponding to the different HCVgenotypes.

The data in FIG. 54 can be more readily interpreted by using a firstderivative plot of the same data. FIG. 55 shows the data in FIG. 54 as afirst derivative plot. The peak of each curve represents the T_(m) ofthe hybridization complex at those particular hybridization conditions.As can be seen, the T_(m) for each HCV genotype can be easilydistinguished on the graph. Thus, the soluble quencher thiazine dyeazure B can be successfully used in a melting curve Tm determinations.

Example 20 Demonstration of Nucleic Acid Duplex Stabilization in thePresence of Thiazine Dyes Using an HCV Model System

The present example illustrates the duplex stabilization properties ofthiazine dyes. An HCV probe and HCV synthetic templates are used in theexperimental system, and duplex stabilization is demonstrated bymeasuring the Tm of the various hybridization complexes that are formed.

Melting curve reactions were established using the single-label probe(SEQ ID NO: 14) and HCV type 1a/b synthetic template (SEQ ID NO: 15) asshown in FIG. 56. This analysis used the same methodologies as describedin the Example above. This particular combination of probe and HCVgenotype 1a/b template produces a perfect alignment (no mismatches).These melting analysis reactions alternatively contained four increasingconcentrations of the thiazine dye methylene blue ranging from 10-40μg/mL. The melting data is shown in FIG. 56 as a first derivative plotof fluorescence versus temperature. The results of the four separateexperiments are overlaid on the same graph. A representative set of datais shown. As can be seen in the figure, the increasing concentration ofmethylene blue resulted in increased stability of the perfect matchduplexes, reflected in the higher Tm values as the concentration ofmethylene blue was increased.

Three additional experiments were run where the duplexes contain one,two or three mismatches, and the effects of methylene blue on duplexstability was assessed. In the first of these experiments, melting curvereactions were established using the same HCV genotyping probe as usedin FIG. 56, and an HCV synthetic template corresponding to HCV genotype6 (SEQ IN NO: 20), as shown in FIG. 57. This particular combination ofprobe and genotype 6 template produces a nucleic acid duplex containingone mismatch position. As in the previous experiment, the meltingreactions contained alternatively four increasing concentrations of thethiazine dye methylene blue ranging from 10-40 μg/mL. The melting datais shown in FIG. 57 as a first derivative plot of fluorescence versustemperature. The results of the four separate experiments are overlaidon the same graph. A representative set of data is shown. As can be seenin FIG. 57, the increasing concentration of methylene blue resulted inincreased stability of the duplexes containing one mismatch, reflectedin the higher Tm values as the concentration of methylene blue wasincreased.

Similarly, melting curve reactions were also established using the sameHCV genotyping probe and an HCV synthetic template corresponding to HCVgenotype 5 (SEQ IN NO: 19), as shown in FIG. 58. This combination ofprobe and genotype 5 template produces a nucleic acid duplex containingtwo mismatch positions. The melting reactions contained alternativelyfour increasing concentrations of the thiazine dye methylene blue. Themelting data is shown in FIG. 58 as a first derivative plot offluorescence versus temperature. The results of the four separateexperiments are overlaid on the same graph. A representative set of datais shown. As can be seen in FIG. 58, the increasing concentration ofmethylene blue resulted in increased stability of the duplexescontaining two mismatches, reflected in the higher Tm values as theconcentration of methylene blue was increased.

Melting curve reactions were also established with HCV probe andtemplate that resulted in nucleic acid duplexes containing threemismatch positions, as shown in FIG. 59. These reactions used an HCVsynthetic template corresponding to HCV genotype 2a/c (SEQ ID NO: 16).The melting reactions contained alternatively four increasingconcentrations of the thiazine dye methylene blue. The melting data isshown in FIG. 59 as a first derivative plot of fluorescence versustemperature. where the results of the four separate experiments areoverlaid on the same graph. A representative set of data is shown. Ascan be seen in FIG. 59, the increasing concentration of methylene blueresulted in increased stability of the duplexes containing the threemismatches, reflected in the higher Tm values as the concentration ofmethylene blue was increased.

It is significant to note that the degree of stabilization is morepronounced with increasing concentrations of methylene blue, andfurthermore, duplexes that containing increasing numbers of mismatchesshow larger degrees of stabilization as measured by Tm. For example,with no mismatches present in a duplex (FIG. 56), the difference in Tmvalues when using 10 μg/mL methylene blue versus 40 μg/mL methylene blueis 2.0° C. However, with three mismatches present in a duplex (FIG. 59),the difference in Tm values when using 10 μg/mL methylene blue versus 40μg/mL methylene blue is much more pronounced with a 12.4° C. spreadbetween those reaction conditions. Duplexes having one, and twomismatches present (FIGS. 57 and 58) show intermediate degrees of duplexstabilization.

Example 21 Nucleic Acid Duplex Stabilization in the Presence of ThiazineDyes Using Multiple HCV Template Targets

The duplex stabilization feature of thiazine dyes is further illustratedin the bar graph provided in FIG. 60. This bar graph provides a summaryof Tm determinations using the HCV probes indicated with the varioussynthetic nucleic acids having nucleotide sequences corresponding to theHCV genotypes shown. This analysis examined the effects of methyleneblue, where alternatively no methylene blue, 10 μg/mL methylene blue or20 μg/mL methylene blue were used in the melting curve analysis.

These determinations were done using one of two different methodologies.In one set of experiments, Tm determinations were made in the absence ofnew methylene blue. In that case, a single-labeled FAM probe would beineffective in the Tm determination, because there is not a suitabledonor/quencher pair present to monitor duplex formation/dissociation. Inthat case, a probe was synthesized without a FAM label (SEQ ID NO: 22),and the melting curve and Tm determination were accomplished byincluding SYBR® Green in the reaction. SYBR® Green staining is specificfor double stranded DNA, and so is an effective monitor for duplexassociation/dissociation. Alternatively, when methylene blue was presentin the melting reactions, a single-labeled FAM probe (SEQ ID NO: 14) wasused as previously described. In these experiments, the nucleotidesequences of the two different probes were identical; the onlydifference between the two probes was the absence/presence of the FAMlabel.

The resulting duplexes contained varying numbers of nucleotidemismatches, as shown below:

HCV Genotype Number of Nucleotide Mismatches Present Template when in aDuplex with the Probe 1a/b 0 2a/c 3 2b 4 3a 5 4 3 5 2 6 1

The results of this type of analysis using seven different synthetictemplates corresponding to various HCV genotypes/subtypes, aresummarized in FIG. 60, and demonstrate the general nature of the duplexstabilization effect. A representative set of data is shown. This datais also summarized in the table below.

Genotype Treatment 3a 2b 2a/c 4 5 6 1a/b  0 μg/mL new 32 36 39 32 48.956.8 64.5 methylene blue + unlabeled probe + SYBR ® Green 10 μg/mL new34.2 40 41.1 45.6 53.7 58 66.9 methylene blue + labeled probe 20 μg/mLnew 38.2 42.2 47 47.7 54 61.2 68.2 methylene blue + labeled probe

Two distinct trends can be observed in this figure, illustrating theduplex stabilizing effects of the methylene blue. First, in the analysisof any one HCV genotype, there is an elevation in duplex stability withthe addition of increasing concentrations of methylene blue. This istrue for both perfectly matched duplexes (genotype 1a/b) as well asduplexes containing one or more mismatches (all other genotypes).Second, those duplexes that contain larger numbers of mismatchesgenerally showed the greatest degree of stabilization (as measured bythe changes in the Tm) with the addition of methylene blue to themelting analysis. For example, duplexes containing HCV genotype 1a/b(perfect match, no mismatches) showed only a slight elevation inexperimentally observed Tm value with the addition of methylene blue. Incontrast, duplexes having one or more mismatched nucleotide positionsshowed greater degrees of improved duplex stability with the addition ofnew methylene blue. Similar effects were also observed when using thethiazine dye methylene blue.

It is significant to point out that in the case of the experimentssummarized in FIG. 60, the methylene blue is serving two functions. Inthe case where a single FAM-labeled probe is used in the meltinganalysis, the new methylene blue is first serving as a soluble quencherwith the FAM-labeled probe in order to monitor duplexassociation/dissociation. Second, as illustrated herein, the newmethylene blue is acting to stabilize duplexes, and most significantly,duplexes that contain nucleotide mismatches. In this comparison, thedata for the zero dye (new methylene blue) controls is obtained by usingunlabeled probe and SYBR Green detection for the Tm determination.

Example 22 Demonstration of Single Nucleotide Mismatch Stabilization inthe Presence of Thiazine Dyes

The present example further illustrates single nucleotide mismatchstabilization properties of thiazine dyes by demonstrating a stabilizinginfluence on each of eight different types of nucleotide mismatches. AnHCV probe and synthetic templates are used in the experimental system,and single nucleotide mismatch stabilization is demonstrated bymeasuring the Tm of the various hybridization complexes that are formed.

Melting curve reactions were established using the single-labeled FAMprobe shown in FIG. 52A and synthetic templates that were engineered tocontain various single base mismatches when hybridized with this probe.Note that these engineered templates do not correlate with anyparticular HCV genotypes, and were constructed for illustrative purposesonly. These engineered templates are shown below. The nucleotideposition that is mismatched when annealed to the probe is shown inlowercase. One of the templates was designed with no mismatches.

SEQ ID Synthetic Template NO: CCGGTCGTCCTGGCAATTCCG 26CCGGTCGTCCcGGCAATTCCG 27 CCGGTCGTCCgGGCAATTCCG 28 CCGGTCGTCCaGGCAATTCCG29 CCGGTCGTCCTGGCcATTCCG 30 CCGGTCGTCCTGGCgATTCCG 31CCGGTCGTCCTGGCtATTCCG 32 CCGGTCGTCCTcGCAATTCCG 33 CCGGTCGTCgTGGCAATTCCG34

These combinations of probe and template produced the single nucleotidemismatches shown in the FIG. 61. One hybridization template was includedthat produced no mismatches with the probe (i.e., an A:T perfect match).Each melting analysis reaction contained alternatively 10 μg/mLmethylene blue or 40 μg/mL methylene blue.

This Tm melting data is summarized in the bar graph in FIG. 61. Arepresentative set of data is shown. Also indicated on the graph is thepredicted Tm of the respective hybridization complexes (in the absenceof methylene blue). These calculated values were derived from Visual OMPsoftware estimates (DNA Software, Inc., Ann Arbor, Mich.).

As can be seen in the figure, the addition of methylene blue to themelting reactions significantly stabilized the mismatched duplexes, asdetermined by their respective Tm values compared to the predicted Tmvalues in the absence of methylene blue. Furthermore, the addition of 40μg/mL methylene blue appeared to be more effective than 10 μg/1 mLmethylene blue at stabilizing the duplexes. These methylene bluestabilizing effects also were also observed in the perfect match duplex.These data demonstrate that the duplex stabilization effect is notlimited to any particular mismatch types, and is a general phenomenon.In some cases, the degree of stabilization is dependent on the mismatchtype.

Example 23 Demonstration of Improved Subtype Detection in the Presenceof Thiazine Dyes Using an HIV Model System

The present example illustrates the benefits of the duplex stabilizationproperties of thiazine dyes in the amplification and detection of viraltargets. Because of the ability of the thiazine dyes to stabilizemismatched duplexes, it is shown herein that the detection sensitivityof polymorphic subtypes can be greatly improved due to improvedamplification and/or detection efficiencies. In contrast to the previousexample, this example used C_(T) values of various TaqMan amplificationreactions to demonstrate the benefits of enhanced duplex stability. HIVamplification primers, an HIV double-labeled 5′-nuclease quantitationprobe and HIV synthetic templates were used in the experimental modelsystem, as provided in FIG. 62. Beneath the primer and probe sequencesin FIG. 62, the corresponding homologous domains from known HIV isolatesare provided, with the variable positions indicated.

Single tube RT-PCR amplification reactions for the real-timequantitation of amplicon products were established using theamplification primers as shown below:

Amplifi- SEQ cation ID primer Sequence NO SK145BUAGTGGGGGGACATCAAGCAGCCATGCAA-tBuBndA 23 GAG152BUGGTACTAGTAGTTCCTGCTATGTCACTTC-tBuBndA 24 where tBuBndA= N6-t-butylbenzyl-dA

The amplification reactions also included the double-labeled 5′-nucleasequantitation probe GAG108FBHQ29I having the sequence:

(SEQ ID NO: 25) FAM-TCTGCAGCT BHQ2 TCCTCATTGATGGTATCTTTTA-PO₄where FAM is the fluorescent label, PO₄ is a terminal phosphate and BHQ2is the black hole quencher (BHQ™)-2. The amplification reactions alsoincluded synthetically produced HIV RNA molecules produced by in vitrotranscription of subcloned isolated HIV genetic material and purified byoligo-dT-sepharose chromatography. One million copies of the specifiedRNA transcript were used in each reaction. The PCR reaction used thefollowing cycling program:

50° C./5 min; 59° C./30 min; 95° C./2 min; 95° C.→58° C. (2 cycles); 91°C.→58° C. (60 cycles)

In a first experiment, an HIV RNA amplification (RT-PCR) quantitationusing the SK145BU and GAG152BU amplification primers and theGAG108FBHQ29I 5′-nuclease quantitation probe was established. Theexperimental results are provided in FIG. 63. Various HIV RNA templates(10⁶ copies each) were used in separate amplification reactions, asindicated. The numbers of nucleotide mismatches in the forward primer,reverse primer and the 5′-nuclease probe are shown for each HIV subtypetested in the table below. No thiazine dye is present in the reactions.A representative set of data is shown. Also indicated are the C_(T)numbers obtained for each HIV subtype. As seen in FIG. 63 and the table,each HIV genotype tested has a distinct C_(T) number.

The HIV RNA amplification quantitation analysis provided in FIG. 63 wasrepeated in the experiment shown in FIG. 64, with the exception that thereactions were supplemented with 50 μg/mL of new methylene blue. As canbe seen, the addition of the new methylene blue resulted in improvedlevels of detection, where the C_(T) values are lowered with theaddition of the thiazine dye. These results are summarized below:

No. of duplex mismatches in: C_(T) value C_(T) value with HIV forprimer/rev primer/ without new 50 μg/mL Genotype nuclease probemethylene blue new methylene blue  101-15 0/0/3 ND ND 105-1 0/0/1 24.824.2 106-1 2/1/2 27.7 25.2 108-3 1/1/3 28.3 27.2 109-1 2/2/2 28.2 27.1110-5 6/0/1 33.0 23.9

As shown in FIGS. 63 and 64, and summarized in table above, the duplexstabilization properties of thiazine dyes are demonstrated in this modelsystem, likely through stabilizing both the primer-template duplex aswell as the amplicon-nuclease probe duplex. This stabilization leads tobetter detection sensitivity for polymorphic subtypes.

Example 24 Demonstration of Dose-Dependent Nucleic Acid DuplexStabilization in the Presence of Varying Concentrations of Thiazine DyesUsing an HIV Model System

The present example illustrates the duplex stabilization properties ofthiazine dyes, where the dye is employed at a range of concentrations.This example uses the same HIV experimental model system and reagents asdescribed in Example 23.

Amplification reactions for the real-time quantitation of HIVamplification products were established using the HIV amplificationprimers SK145BU (SEQ ID NO: 23) and GAG152BU (SEQ ID NO: 24) and the5′-nuclease quantitation probe GAG108FBHQ29I (SEQ ID NO: 25). Theamplification reactions in this Example targeted the HIV genotype 110-5synthetic RNA template (10⁶ copies). This particular combination of HIVgenotype, amplification primers and nuclease probe results in sixmismatches under the forward primer and one mismatch under the5′-nuclease quantitation probe.

The amplification and quantitation reactions were alternativelysupplemented with various concentrations of new methylene blue from10-50 μg/mL. One reaction without any new methylene blue was also run.Results are displayed as amplicon growth curves and C_(T) values. Theresults are provided in FIG. 65. A representative set of data is shown.As clearly seen in the figure, the addition of new methylene blueresults in increased sensitivity of the amplification and quantitationassay, as evidenced by the decreased C_(T) values with increasingthiazine dye concentration. This is likely through stabilizing both theprimer-template duplex as well as the amplicon-nuclease probe duplex.This data clearly shows the beneficial effect of new methylene blue onthe detection sensitivity of this HCV subtype as a function ofincreasing concentration of the dye.

Example 25 Demonstration of Nucleic Acid Duplex Stabilization byThiazine Dyes Using SYBR® Green Amplicon Detection

The present example illustrates the duplex stabilization properties ofthiazine dyes, where the model system uses SYBR® Green to monitoramplicon accumulation. Amplification reactions for the real-timequantitation of HIV amplification products were established using theHIV amplification primers SK145BU (SEQ ID NO: 23) and GAG152BU (SEQ IDNO: 24). The amplification reactions in this Example targeted the HIVgenotype 110-5 synthetic RNA template (10⁶ copies). All the reactionswere supplemented with SYBR® Green to monitor accumulation of thedouble-stranded amplicon product. A 5′-nuclease quantitation probe wasnot used.

The amplification reactions were alternatively supplemented with either30 μg/mL or 50 μg/1 mL new methylene blue. A reaction was also run inthe absence of new methylene blue. When no new methylene blue was usedin the reaction, a 1:10,000 dilution (1× concentration) of SYBR® Greenwas used. When the 30 and 50 μg/mL new methylene blue were used, adilution of 1:2,500 of SYBR® Green was (4× concentration). SYBR® Greenfluorescence was measured at the same wavelength as the FAM label. Theaddition of new methylene blue to the reactions had the effect ofreducing the fluorescence of the SYBR® Green emission. To compensate forthis, the reactions containing new methylene blue used the higherconcentration of SYBR® Green. The increased concentration of SYBR® Greenis known to have a negative influence on the amplification efficiency.However, in spite of this detrimental effect, the beneficial effect ofthe new methylene blue on duplex stabilization is clearly seen.

The results of this assay are shown in FIG. 66. Results are displayed asamplicon growth curves and C_(T) values. A representative set of data isshown. As clearly seen in the figure, the addition of new methylene blueresults in improved sensitivity of the amplification and quantitationassay, as evidenced by the decreased C_(T) values with increasingthiazine dye concentration. Presumably, the thiazine dye is stabilizingthe interaction of the amplification primers with the HIV targettemplate. Furthermore, this Example illustrates that a SYBR® Greendetection system can be used in conjunction with a thiazine dye for thestabilization of DNA duplexes.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All scientific publications, patentpublications of any type, issued patents, pending patent applications,and/or other documents cited in this application are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent, patent application, and/or otherdocument were individually indicated to be incorporated by reference forall purposes.

What is claimed is:
 1. A method for producing a stabilized nucleic acidduplex, the method comprising: (a) providing (i) a sample containing orsuspected of containing a target nucleic acid molecule; (ii) anoligonucleotide complementary or partially complementary to said targetnucleic acid molecule; and (iii) at least one soluble thiazine dyepresent at a concentration effective to stabilize a duplex formedbetween said target nucleic acid molecule and said oligonucleotide,wherein said target nucleic acid, said oligonucleotide and said thiazinedye are all in solution in free form; and (b) either (i) annealing saidtarget nucleic acid and said oligonucleotide in the presence of saidthiazine dye; or (ii) annealing said target nucleic acid and saidoligonucleotide, followed by admixing with said thiazine dye; underconditions wherein base-pairing occurs to produce at least onestabilized nucleic acid duplex.
 2. The method of claim 1, whereinstability of said nucleic acid duplex of (b) is improved compared tostability of a nucleic acid duplex comprising said target nucleic acidand said oligonucleotide in the absence of said thiazine dye.
 3. Themethod of claim 1, further comprising: (c) detecting said stabilizednucleic acid duplex under conditions that provide improved stability. 4.The method of claim 1, wherein said stabilized nucleic acid duplexcomprises one or more nucleobase mismatches.
 5. The method of claim 1,wherein said providing said target nucleic acid molecule and saidoligonucleotide comprises providing one nucleic acid molecule, whereinsaid nucleic acid molecule is a molecular beacon.
 6. The method of claim1, wherein said oligonucleotide is effective to prime a nucleic acidextension reaction when annealed to said target nucleic acid.
 7. Themethod of claim 1, wherein step (a) further comprises providing a secondoligonucleotide complementary or partially complementary to said targetnucleic acid molecule, each of said oligonucleotides being effective toprime a nucleic acid extension reaction when annealed to said targetnucleic acid.
 8. The method of claim 1, wherein step (a) furthercomprises providing a labeled oligonucleotide complementary or partiallycomplementary to said target nucleic acid molecule.
 9. The method ofclaim 1, wherein said target nucleic acid molecule is an amplicon. 10.The method of claim 1, wherein said oligonucleotide comprises one ormore naturally-occurring nucleotides, modified nucleotides, nucleotideanalogs, one or more unnatural bases, unnatural internucleotidelinkages, unnatural nucleotide backbones, or any combination thereof.11. The method of claim 1, wherein said thiazine dye is selected frommethylene blue, methylene green, thionin, sym-dimethylthionin, toluidineblue O, new methylene blue, methylene violet bernthsen, azure A, azureB, azure C and 1,9-dimethylmethylene blue.
 12. The method of claim 1,wherein said at least one thiazine dye is present at a concentration ofat least 10 μg/mL.
 13. The method of claim 1, wherein said annealingcomprises annealing in the presence of a thiazine dye at a concentrationbetween about 10 μg/mL and 50 μg/mL.
 14. The method of claim 1, whereinsaid annealing comprises annealing in the presence of a thiazine dye ata concentration of about 40 μg/mL.